PHYSIOLOGICAL REVIEWS

Vol. 78, No. 2, April 1998

Printed in U.S.A.

The Free Radical Theory of Aging Matures

KENNETH B. BECKMAN AND BRUCE N. AMES

Department of Molecular and Cell Biology, University of California, Berkeley, California

I. Introduction 548
II. An Overview of the Free Radical Theory of Aging 549
A. Origins of the free radical theory 549
B. Sources of oxidants 549
C. Targets of oxidants 552
D. Antioxidant defenses 553
E. Repair of oxidative damage 554

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F. Synthesis: interaction of oxidant generation, oxidative damage, and repair 554
III. Refinements and Corollaries of the Free Radical Theory 554
A. Oxidants and evolutionary theories of aging 554
B. Oxidants and the somatic mutation theory of aging 555
C. Oxidants and mitochondrial theories of aging 556
IV. Oxidative Phenomenology: Age-Associated Trends 556
A. Accumulation of oxidative end products 556
B. Steady-state levels of oxidative modification 557
C. Oxidative depletion of biochemical pools 559
D. Age-associated trends in antioxidant defenses and repair 559
E. Age-associated trends in oxidant generation 560
V. Interspecies Comparisons 561
A. Oxidative damage and maximum life span potential 561
B. Antioxidant defenses and maximum life span potential 561
C. Generation of reactive oxygen species and maximum life span potential 562
VI. Dietary Restriction 562
VII. Manipulation of Rate of Living 563
VIII. Manipulation of Oxygen Tension 563
IX. Supplementation With Dietary Antioxidants 563
X. Administration of Pharmacological Antioxidants 564
XI. In Vitro Senescence and Oxidants 564
XII. Classical and Population Genetics 565
A. Caenorhabditis elegans genetics 565
B. Drosophila genetics 566
C. Rodent genetics 567
XIII. Molecular Genetics 567
A. Oxidants and nuclear somatic mutations 567
B. Oxidants and mitochondrial somatic mutations 567
XIV. Transgenic Organisms 568
A. Transgenic Drosophila 568
B. Transgenic mice 568
XV. Sporadic Degenerative Diseases 569
XVI. Inherited Degenerative Diseases 569
XVII. Epidemiology of Oxidants and Antioxidants 569
XVIII. Summary 570
A. Are oxidants responsible for aging? 570
B. Nitric oxide 571
C. Oxidants and apoptosis 571
D. Regulatory roles for oxidants and antioxidants: signal transduction 571

Beckman, Kenneth B., and Bruce N. Ames. The Free Radical Theory of Aging Matures. Physiol. Rev. 78: 547
581, 1998.The free radical theory of aging, conceived in 1956, has turned 40 and is rapidly attracting the interest
of the mainstream of biological research. From its origins in radiation biology, through a decade or so of dormancy
and two decades of steady phenomenological research, it has attracted an increasing number of scientists from an
expanding circle of fields. During the past decade, several lines of evidence have convinced a number of scientists

0031-9333/98 $15.00 Copyright q 1998 the American Physiological Society 547

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548 KENNETH B. BECKMAN AND BRUCE N. AMES Volume 78

that oxidants play an important role in aging. (For the sake of simplicity, we use the term oxidant to refer to all
reactive oxygen species, including O0
2 j, H2O2 , and jOH, even though the former often acts as a reductant and
produces oxidants indirectly.) The pace and scope of research in the last few years have been particularly impressive
and diverse. The only disadvantage of the current intellectual ferment is the difficulty in digesting the literature.
Therefore, we have systematically reviewed the status of the free radical theory, by categorizing the literature in
terms of the various types of experiments that have been performed. These include phenomenological measurements
of age-associated oxidative stress, interspecies comparisons, dietary restriction, the manipulation of metabolic
activity and oxygen tension, treatment with dietary and pharmacological antioxidants, in vitro senescence, classical
and population genetics, molecular genetics, transgenic organisms, the study of human diseases of aging, epidemio-
logical studies, and the ongoing elucidation of the role of active oxygen in biology.

I. INTRODUCTION that fruit flies and nematodes are amenable to the study
of aging. Also, medical researchers investigating human
The study of aging, by nature multidisciplinary, has diseases of aging, such as Alzheimers disease (AD) and
been characterized by a dizzying variety of theories, a inherited progerias, have overcome long-standing road-

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huge phenomenological literature, and the absence of
firmly established primary causes. The diverse life histo-
ries of animal species, which manifest aging in very differ-
ent ways, has been an obstacle to testing unified theories.
For experimental gerontology to provide more than a cat-
alog of age-related changes, it has been necessary for
biologists to define the alterations that are common to
most old cells, tissues, and animals, while simultaneously
respecting that there is not a single phenomenon of aging
or a single cause. This has taken some time, and from the
outside it may have appeared that the field has been mired
in phenomenology. Perhaps for this reason, the study of
aging was until recently avoided by molecular biologists,
who naturally favored clear-cut phenomena. As the molec-
ular details of development, cancer, and immunology
yielded to modern tools during the 1970s and 1980s, the
field of aging lagged, and mechanisms responsible for
aging failed to emerge.
Throughout this time, though, there has never been
a shortage of unified theories attempting to reduce aging
to something more tractable. In fact, gerontologists have
been prolific in this regard (83, 205). Whereas some re-
searchers have believed that a small number of random,
deleterious mechanisms could explain degenerative se-
nescence, others have opted for theories of pro-
grammed aging, in which senescence is the final destina-
tion in a developmental pathway. In the course of these
debates, a number of scientists have rallied around a set
of ideas called the free radical theory of aging: loosely, the
belief that damage by reactive oxygen species is critical in
determining life span. This theory inspired many experi-
ments in which evidence of oxidative damage in aged
animals was sought.
In the last 10 years or so, the nature of aging research
has changed dramatically; one might say that the field has
entered early adulthood. The tools of molecular biology
are now sophisticated and accessible enough that re-
searchers within gerontology have adopted them. At the
same time, molecular biologists situated on the edge of
aging research have made inroads and have discovered
blocks.
It has been gratifying, therefore, that many of the
preliminary studies in what might be called molecular
gerontology lend credibility to the free radical theory.
Results from disparate experimental systems have re-
cently shown that oxygen radicals play a role in degenera-
tive senescence, and the pace of discoveries is quickening.
The likely result of this collision of scientific approaches
will be the unraveling of the physiological tangle of aging,
and it seems safe to say that one of the important knots
will turn out to be oxidative stress.
However, there is a danger that in the excitement
of theoretical confirmation, certain nuances are lost. For
instance, the revelation that oxygen radicals may be in-
volved in neurodegeneration does not mean that oxidative
stress determines life span. The free radical theory has
sought not only to explain the mechanisms of degenera-
tive senescence, but it has also attempted to explain dif-
ferences between species life spans in terms of oxidants.
So, although many recent studies indicate that oxygen
radicals play some kind of role in aging, only a small
number of these support the more ambitious version of
the free radical theory. On the other hand, there is no
reason to cling to such a stringent version of the free
radical theory, and it is becoming apparent that whether
or not they determine life span, oxygen radicals are cer-
tainly important players in agings pathophysiology. In
other words, the scope of the free radical theory of aging
should include aging-associated oxidative stress in gen-
eral, rather than limiting itself to those oxidative events
that may determine life span. In fact, many current articles
indicate that such a blurring of distinctions has already
occurred and that as it is commonly used, free radical
theory encompasses a broad set of ideas. Therefore, our
first purpose is to delineate these different conceptions
of the free radical theory, as a prerequisite to its critical
evaluation.
Because of the recent popularity of free radical re-
search, a large number of reviews have addressed various
aspects of the interplay between oxidants and aging (6,

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April 1998 FREE RADICAL THEORY OF AGING
15, 18, 33, 51, 57, 58, 60, 65, 82, 85, 87, 103, 106, 123126,
130, 161, 166, 196, 203, 227, 257, 273, 275, 288, 293, 297,
307, 312, 315, 335, 338, 348, 353, 357). Rather than merely
updating this literature, our aim is to provide a systematic
categorization of the types of experiments that have been
performed. The phenomenon and study of aging are in-
credibly diverse, encompassing organisms from rotifers
to mammals and techniques from physiology to genetics.
Although it is precisely the broad sweep of evidence that
lends the free radical theory its appeal, the menagerie of
animals and techniques sometimes obscures the logic. By
breaking the literature down into smaller pieces (a prac-
tice we find necessary ourselves), we hope to make it
easier for readers to judge the theory. Moreover, by impos-
ing a structure, we aim to highlight novel and definitive
approaches, because it is these that will replace the phe-
nomenology of past decades.
In this review, then, we briefly outline the evolution
of the free radical theory and then delineate the different
areas of evidence. We focus on recent experiments and
point to the areas that we feel are most likely to provide
future insights. The way in which we have categorized
the literature is outlined in the table of contents (sects.
IV-XVII) and in Table 1. Although any such system is some-
what arbitrary, we hope that ours will make it easier both
to assimilate the existing literature and to envision future
experiments. In writing a review on as broad a topic as
the free radical theory, we have been forced to limit both
the content and the number of references cited. Although
we have done our best to include recent work, omissions
were inevitable. We apologize to all authors whose work
we have not managed to include, and direct readers to
other recent reviews for material we have left out.
II. AN OVERVIEW OF THE FREE RADICAL
THEORY OF AGING
A. Origins of the Free Radical Theory
In 1956, Denham Harman suggested that free radicals
produced during aerobic respiration cause cumulative ox-
idative damage, resulting in aging and death. He noted
parallels between the effects of aging and of ionizing radi-
ation, including mutagenesis, cancer, and gross cellular
damage (120, 128). At the time, it had recently been dis-
covered that radiolysis of water generates hydroxyl radi-
cal (jOH) (319), and early experiments using paramag-
netic resonance spectroscopy had identified the presence
of jOH in living matter (45). Harman (120) therefore hy-
pothesized that endogenous oxygen radical generation oc-
curs in vivo, as a by-product of enzymatic redox chemis-
try. He ventured that the enzymes involved would be those
involved in the direct utilization of molecular oxygen,
particularly those containing iron. Finally, he hypothe-
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549
sized that traces of iron and other metals would catalyze
oxidative reactions in vivo and that peroxidative chain
reactions were possible, by analogy to the principles of
in vitro polymer chemistry. All of these predictions have
been confirmed during the past 40 years.
The theory gained credibility with the identification in
1969 of the enzyme superoxide dismutase (SOD) (204),
which provided the first compelling evidence of in vivo
generation of superoxide anion (O20j), and from the subse-
quent elucidation of elaborate antioxidant defenses (351).
The use of SOD as a tool to locate subcellular sites of
O20j generation led to a realization that buttressed the free
radical theory, namely, that mitochondria are a principal
source of endogenous oxidants (37). Gerontologists had
long observed that species with higher metabolic rates
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have shorter maximum life span potential (MLSP); they
age faster (268). In fact, it had been proposed at the turn
of the century that energy consumption per se was respon-
sible for senescence, a concept referred to as the rate of
living hypothesis (246, 268, 295). The realization that en-
ergy consumption by mitochondria may result in O20j pro-
duction linked the free radical theory and the rate of living
theory irrevocably: a faster rate of respiration, associated
with a greater generation of oxygen radicals, hastens aging.
By now, the two concepts have essentially merged.
B. Sources of Oxidants
Ground-state diatomic oxygen (3Sg0O2 or more com-
monly, O2), despite being a radical species and the most
important oxidant in aerobic organisms, is only sparingly
reactive itself due to the fact that its two unpaired elec-
trons are located in different molecular orbitals and pos-
sess parallel spins. As a consequence, if O2 is simultane-
ously to accept two electrons, these must both possess
antiparallel spins relative to the unpaired electrons in O2 ,
a criterion which is not satisfied by a typical pair of elec-
trons in atomic or molecular orbitals (which have oppo-
site spins according to the Pauli exclusion principle). As
a result, O2 preferentially accepts electrons one at a time
from other radicals (such as transition metals in certain
valences). Thus, in vivo, typical two- or four-electron re-
duction of O2 relies on coordinated, serial, enzyme-cata-
lyzed one-electron reductions, and the enzymes that carry
these out typically possess active-site radical species such
as iron. One- and two-electron reduction of O2 generates
O20j and hydrogen peroxide (H2O2), respectively, both of
which are generated by numerous routes in vivo, as dis-
cussed below. In the presence of free transition metals
(in particular iron and copper), O20j and H2O2 together
generate the extremely reactive hydroxyl radical (jOH).
Ultimately, jOH is assumed to be the species responsible
for initiating the oxidative destruction of biomolecules.
In addition to O20j, H2O2 , and jOH, two energetically ex-
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550 KENNETH B. BECKMAN AND BRUCE N. AMES Volume 78

TABLE 1. The strengths and weaknesses of approaches to the testing of the free radical theory of aging
Experimental Approach
(Review Section) Strengths of the Approach Weaknesses of the Approach

Oxidative phenomenology Simplicity; large existing data set; elucidation of the
(Sect. IV) basic biochemistry of oxidative stress; technical
foundation for other approaches; negative result
may be instructive
Interspecies comparisons Specific testable predictions; deviations from
(Sect. V) predictions may refine theory; use of different
species avoids conclusions that are species or
strain specific; negative results may be instructive
Dietary restriction Very well established model of life span extension;
(Sect. VI) straightforward methodology; probably relevant to
human aging and cancer
Manipulation of rate of A direct test of rate of living theory, with specific
living testable predictions; straightforward methodology
Results are merely correlative (oxidative damage
may be a consequence of aging); negative
results, considered uninteresting, may fail to
appear in the literature
Logistical problems in animal handling;
quantitative comparisons complicated by
qualitative interspecies differences in oxidative
defenses and/or repair
Results are somewhat correlative (alterations in
oxidative stress may be consistent with but
incidental to a more fundamental cellular
switch)
To date, limited to relatively simple organisms,
such as invertebrates

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(Sect. VII)
Manipulation of oxygen A direct test of rate of living theory, with specific Results hard to interpret. Are positive results
concentration testable predictions; straightforward methodology (life span attenuation by hyperoxia, extension
(Sect. VIII) by hypoxia) relevant to normoxic aging? Not
applicable to most mammals
Supplementation with (Potentially) a test of free radical theory Results hard to interpret, due to unknown fates
dietary antioxidants of supplements, potential adverse effects,
(Sect. IX) complexity of overall antioxidant defenses
Administration of (Potentially) a test of free radical theory Results hard to interpret, due to unknown fates
pharmacological of supplements, potential adverse effects,
antioxidants complexity of overall antioxidant defenses
(Sect. X)
In vitro senescence A very well-established model system; relevance to Relevance to aging in vivo not yet clear
(Sect. XI) human cancer (and perhaps to degenerative
senescence in vivo)
Classical and population Awesome power of genetics; very well established Currently limited to invertebrate model
genetics methods linked to a vast body of species-specific organisms (yeast, C. elegans, Drosophila)
(Sect. XII) information; concurrent genome projects may
forge interspecies links between gerontogenes
Molecular genetics Powerful techniques for measuring mutational events In addition to causing cancer, physiological
(Sect. XIII) (quantitatively and qualitatively); mutational relevance of somatic mutations (and other
fingerprinting to identify oxidative stress; simple genetic abnormalities) is not obvious
methods applicable to wide array of species
Transgenic organisms Immensely powerful tools; a direct test of free Somewhat expensive and time consuming;
(Sect. XIV) radical theory; interbreeding of transgenic animals dynamic nature of oxidative stress may
for sophisticated in vivo research; established complicate interpretations of phenotypes
methods for widely divergent animals
(Caenorhabditis elegans, Drosophila, mice)
Sporadic degenerative Results (if positive) provide mechanistic insights into Lack of animal models; disease may be of
diseases the biology of oxidative pathophysiology and strictly human relevance (not a general
(Sect. XV) evidence of etiological relevance phenomenon of aging)
Inherited degenerative Results (if positive) provide mechanistic insights into Usually lack of an animal model; phenomenon
diseases the biology of oxidative pathophysiology and (often severe) may not be of relevance either
(Sect. XVI) evidence of etiological relevance to most humans, nor to aging in general
Epidemiology Large data sets permit subtle specieswide trends to Currently expensive, long term, and difficult
(Sect. XVII) be detected; public health and policy relevance
(preventative medicine); may be facilitated by
sequencing of the human genome

cited species of O2 termed singlet oxygens can result
from the absorption of energy (for instance, from ultravio-
let light). Designated by the formulas 1DgO2 and 1Sg/O2 ,
both of these species differ from the triplet ground state
(3Sg0O2) in having their two unpaired electrons in oppo-
site spins, thereby eliminating the spin restriction of
ground-state O2 and enabling greater reactivity. The chem-
istry of oxygen and its derivatives has been extensively
discussed elsewhere (115, 342). Because all of these spe-
cies (O20j, H2O2 , jOH, 1DgO2 , and 1Sg/O2), by different

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April 1998 FREE RADICAL THEORY OF AGING
routes, are involved in oxygens toxicity, we will collec-
tively refer to them as oxidants.
It is now beyond doubt that oxidants are generated
in vivo and can cause significant harm (20, 37, 60, 91, 112,
351). There are numerous sites of oxidant generation, four
of which have attracted much attention: mitochondrial
electron transport, peroxisomal fatty acid metabolism, cy-
tochrome P-450 reactions, and phagocytic cells (the res-
piratory burst). Before a discussion of the potential con-
tributions of different sources of oxidants, it is worthwhile
briefly to outline them.
In the textbook scheme of mitochondrial respiration,
electron transport involves a coordinated four-electron re-
duction of O2 to H2O, the electrons being donated by NADH
or succinate to complexes I and II, respectively, of the mito-
chondrial electron transport chain (ETC). Ubiquinone (coen-
zyme Q, or UQ), which accepts electrons from complexes
I (NADH dehydrogenase) and II (succinate dehydrogenase),
undergoes two sequential one-electron reductions to ubi-
semiquinone and ubiquinol (the Q cycle), ultimately transfer-
ring reducing equivalents to the remainder of the electron
transport chain: complex III (UQ-cytochrome c reductase),
cytochrome c, complex IV (cytochrome-c oxidase), and fi-
nally, O2 (115). However, it appears mitochondrial electron
transport is imperfect, and one-electron reduction of O2 to
form O0 2 j occurs. The spontaneous and enzymatic dismuta-
tion of O0 2 j yields H2O2 , so a significant by-product of the
actual sequence of oxidation-reduction reactions may be the
generation of O0 2 j and H2O2 .
How much O20j and H2O2 do mitochondria generate?
In classic experiments during the 1970s, measurements of
H2O2 generation by isolated mitochondria indicated that it
is maximal when ADP is limiting and the electron carriers
are consequently reduced (state 4 respiration) (26). Es-
timates of state 4 H2O2 generation by pigeon and rat mito-
chondrial preparations amounted to 12% of total elec-
tron flow (26, 229). One problem with this estimate of
mitochondrial H2O2 generation is its reliance on the use
of buffer saturated with air (20% O2). In vivo, the partial
pressure of O2 is 5%, so these calculations may overesti-
mate the flux of oxidants in vivo. Even disregarding the
use of air-saturated buffer, the initial estimate of percent-
age ETC flux leading to H2O2 can be challenged on the
grounds that in these experiments the concentrations of
substrates fed to mitochondria were higher than occurs
physiologically (118, 146). When H2O2 is measured with
more physiological concentrations, the flux is 10-fold
lower (118), and experiments using subcellular fractions
of SOD-deficient Escherichia coli suggest in vivo leakage
of 0.1% from the respiratory chain (146).
What proportion of mitochondrial H2O2 ultimately de-
rives from ETC O20j generation? Unfortunately, the mea-
surement of O20j generation by intact mitochondria is pre-
vented by the presence of mitochondrial SOD (mSOD).
Therefore, the isolation of submitochondrial particles
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551
from which mSOD has been removed (by sonication of
the intact organelles followed by extensive washing) was
used for the detection of ETC O20j. In these experiments,
stoichiometric estimates of the ratio of O20j generation
(by submitochondrial particles) to H2O2 generation (by
the intact organelles) fell between 1.5 and 2.1 (24, 25, 69,
89, 191); because two O20j molecules dismutate (either
spontaneously or with the help of mSOD) to form one
molecule of H2O2 , such results suggest that virtually all
mitochondrial H2O2 may originate as O20j (27). Moreover,
because most cellular H2O2 originates from mitochondria,
O20j from the ETC may be a cells most significant source
of oxidants (37).
In a recent discussion of the classic in vitro work (88),
some of the original experimenters take issue with the idea
that free O20j exists in mitochondria as a result of normal
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flux through the ETC. They point out that in addition to
having removed mSOD from mitochondria, the sonication
they employed also resulted in the loss of cytochrome c,
which rapidly scavenges O20j in vitro and is present in mito-
chondria at local concentrations from 0.5 to 5 mM. In mito-
chondria, they argue, mSOD and cytochrome c rapidly
scavenge O20j (in the matrix and intermembrane spaces,
respectively). More to the point, the authors stress that
unless the ETC was poisoned with inhibitors such as anti-
mycin A, O20j generation was not detected in their experi-
ments (89, 191). Arguing that mSOD should act to increase
the rate of O20j generation in vivo (by accelerating product
removal by dismutation to H2O2), they suggest that the
actual role of mSOD in vivo may be to increase H2O2 gener-
ation (with O20j as a rapidly consumed intermediate) (88).
Ultimately, there remains a good deal of uncertainty sur-
rounding the mechanisms, quantity, and meaning of mito-
chondrial O20j generation in vivo (228), a mystery which is
deepened by recent reports of enzymatic nitric oxide (NOj)
generation in mitochondria (C. Giulivi and C. Richter, per-
sonal communication). Because O20j and NOj react to form
the oxidant peroxynitrite (ONOO0), mitochondrial O0 2 j gen-
eration may soon need to be considered in the light of its
ability to destroy NOj and form ONOO0, as discussed in
section XVIIIB.
A second source of oxygen radicals is peroxisomal b-
oxidation of fatty acids, which generates H2O2 as a by-prod-
uct. Peroxisomes possess high concentrations of catalase,
so it is unclear whether or not leakage of H2O2 from peroxi-
somes contributes significantly to cytosolic oxidative stress
under normal circumstances. However, a class of nonmuta-
genic carcinogens, the peroxisome proliferators, which in-
crease the number of hepatocellular peroxisomes and result
in liver cancer in rodents, also cause oxidative stress and
damage (7, 157, 177, 230). Interestingly, during the regenera-
tion of the liver after partial hepatectomy, there exist peroxi-
somes that do not stain for catalase activity (232), hinting
that during rapid cell proliferation, oxidant leakage from
peroxisomes may be enhanced.
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552 KENNETH B. BECKMAN AND BRUCE N. AMES
Microsomal cytochrome P-450 enzymes metabolize
xenobiotic compounds, usually of plant origin, by catalyz-
ing their univalent oxidation or reduction. Although these
reactions typically involve NADPH and an organic sub-
strate, some of the numerous cytochrome P-450 isozymes
directly reduce O2 to O20j (105, 168) and may cause oxida-
tive stress. An alternative route for cytochrome P-450-
mediated oxidation involves redox cycling, in which sub-
strates accept single electrons from cytochrome P-450 and
transfer them to oxygen. This generates O20j and simulta-
neously regenerates the substrate, allowing subsequent
rounds of O20j generation (115). Although it is unclear to
what extent cytochrome P-450 side reactions proceed un-
der normal conditions, it is possible that such chronic
O20j generation by cytochrome P-450 is the price animals
pay for their ability to detoxify acute doses of toxins (6).
Finally, phagocytic cells attack pathogens with a mix-
ture of oxidants and free radicals, including O20j, H2O2 ,
NOj, and hypochlorite (38, 220, 262). Although the mas-
sive generation of oxidants by immune cells differs from
the above three sources of free radicals to the extent that
it is the result of pathogenesis, it is nevertheless a normal
and unavoidable consequence of innate immunity.
Chronic inflammation is therefore unique among the en-
dogenous sources of oxidants, because it is mostly pre-
ventable (49, 231, 245).
In addition to these four sources of oxidants, there
exist numerous other enzymes capable of generating oxi-
dants under normal or pathological conditions, often in
a tissue-specific manner (115). To give a single relevant
example, the deamination of dopamine by monoamine
oxidase generates H2O2 , in some neurons, and has been
implicated in the etiology of Parkinsons disease (80).
Finally, the widespread catalytic generation of NOj,
achieved by various isozymes of nitric oxide synthase and
central to processes as diverse as vascular regulation, im-
mune responses, and long-term potentiation, increases
the potential routes for destructive oxidative reactions
(187). The interaction between O20j and NOj results in
ONOO0, which is a powerful oxidant.
As originally articulated by Harman (120), the free
radical theory of aging did not distinguish between these
different sources of oxidants. However, the rate of living
hypothesis clearly singled out mitochondrial O20j and H2O2
generation, since it is the mitochondrial respiration rate
that negatively correlates with MLSP. Also, as many other
established sources of oxidants are tissue specific (associ-
ated with hepatic, neuronal, and other specialized func-
tions), they are less likely to explain aging across a broad
range of species. For this reason, mitochondrial O20j and
H2O2 have captured the lions share of attention. However,
it may turn out that for some age-associated disorders,
nonmitochondrial oxidants are critical. In the expanded
sense of the free radical theory, any oxidants, mitochon-
drial or not, may play a role. Therefore, despite the great
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Volume 78
number of intracellular sources of oxidant that have been
identified in a qualitative way, in terms of ranking their
relative importance, the field is in its infancy.
C. Targets of Oxidants
What are the targets of endogenous oxidants? The
three main classes of biological macromolecules (lipids,
nucleic acids, and proteins) are susceptible to free radical
attack, and there is plentiful evidence that all suffer oxida-
tive damage in vivo. Although it is well beyond the scope
of this review to treat the biochemistry of oxidative dam-
age in any great depth, the area has been expertly re-
viewed (115). A synopsis of the better known pathways
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of oxidative damage, however, is warranted; the most fa-
miliar end products are described here.
The earliest research on the destruction of biological
molecules by oxidants involved lipids (109). Food chem-
ists have long understood that the rancidity of fats results
from peroxidative chain reactions in lipids (autoxida-
tion); a lipid hydroperoxyl radical abstracts a hydrogen
atom from the double bond of a neighboring unsaturated
lipid, forming a hydroperoxide and an alkyl radical, the
latter which combines with O2 to regenerate a lipid hydro-
peroxyl radical capable of initiating another round of oxi-
dation. Ultimately, intramolecular reactions and decom-
position yield cyclic endoperoxides and unsaturated alde-
hydes, the latter of which are reactive and may act as
mutagens (194) or inactivate enzymes (39, 322), or operate
as endogenous fixatives, reacting with proteins and nu-
cleic acids to form heterogeneous cross-links (42). More-
over, a primary effect of lipid peroxidation is decreased
membrane fluidity, which alters membrane properties and
can significantly disrupt membrane-bound proteins (324).
Oxidative damage to nucleic acids includes adducts
of base and sugar groups, single- and double-strand breaks
in the backbone, and cross-links to other molecules. The
spectrum of adducts in mammalian chromatin oxidized in
vitro and in vivo includes more than 20 known products,
including damage to all four bases and thymine-tyrosine
cross-links (70, 71, 113). The electrochemical properties
of the adduct 8-oxo-guanine (oxo8gua) and the deoxy-
nucleoside 8-oxo-2,7-dihydro-2*-deoxyguanosine (oxo8dG),
which have permitted the coupling of extremely sensitive
electrochemical detection to high-performance liquid
chromatography (HPLC), have resulted in hundreds of
studies of its formation, accumulation, and excretion (17).
The identification of specific enzymatic repair of oxidative
lesions has recently provided both proof of the signifi-
cance of oxidative DNA damage as well as tools to manip-
ulate the load of damage in vivo by genetic knockout (17,
23, 78, 192, 266, 291).
The oxidation of proteins is less well characterized,
but several classes of damage have been documented,
pra APS-Phys Rev
April 1998 FREE RADICAL THEORY OF AGING
including oxidation of sulfhydryl groups, reduction of di-
sulfides, oxidative adduction of amino acid residues close
to metal-binding sites via metal-catalyzed oxidation, reac-
tions with aldehydes, protein-protein cross-linking, and
peptide fragmentation (317, 318). A particularly intriguing
recent development has been the realization that a num-
ber of enzymes possessing active-site iron-sulfur clusters
are acutely sensitive to inactivation by O20j (86, 176). For
example, E. coli aconitase is inactivated by O20j with a
rate constant of 109 M01rs01 (95, 96). Mammalian mito-
chondrial aconitase is inactivated in vitro and in vivo by
treatments that increase mitochondrial O20j generation,
such as growth under hyperbaric conditions (97, 98). Be-
cause aconitase participates in the citric acid cycle, its
inhibition would be expected to have pleiotropic effects.
Moreover, the mechanism of aconitase inhibition by O20j
has been demonstrated to involve the release of free iron
from the enzyme (86). Free iron atoms catalytically exac-
erbate oxygen stress (see below), and it has been pro-
posed that superoxides genotoxicity is a function of its
ability to liberate protein-bound iron (159, 184).
Unlike lipids and nucleic acids, proteins represent a
very diverse target for oxidative damage. Although protein
oxidation has been demonstrated at the level of the pep-
tide backbone and amino acids, there has been relatively
little scrutiny of differences between proteins in their sen-
sitivities. A detailed quantitative comparison of bovine
serum albumin and glutamine synthase has shown suscep-
tible residues of the former (methionine and the aromatic
amino acid residues) to be oxidized about twice as fast
as those on the latter, implicating all four levels of protein
structure in relative susceptibility (19). A study of the
oxidation sensitivities of a various cloned K/ channels
from T lymphocytes, cardiac cells, and neurons revealed
that whereas five of the cloned channels were highly sensi-
tive to oxidation, an equal number were resistant (74).
Differential sensitivities raise the possibility that the loss
of homeostasis that is a hallmark of aging could result
from the selective oxidation of proteins.
In the context of aging, a particularly relevant aspect
of oxygens toxicity is its promotion by some metals and
by elevated O2 partial pressure. Iron and copper catalyze
the homolytic cleavage of ROOH (the Fenton reaction),
leading to the generation of jOH (115). It is jOH that is
the most reactive oxidant, reacting at diffusion-limited
rates. The catalytic properties of iron and copper explain
why cells possess metal-chelating proteins such as ferritin
and transferrin, which reduce the concentration of redox-
active metals (114, 211). In humans, the bodys content
of iron increases with age (in men throughout their lives,
and in women after menopause), and it has been sug-
gested that this accumulation may increase the risk of
oxidative damage with age (169, 331). Finally, oxidative
stress in vivo is aggravated by increasing O2 partial pres-
sure, due to a more pronounced flux of mitochondrial
/ 9j08$$ap01 03-24-98 11:38:45
553
O20j (37). Consequently, the manipulation of O2 partial
pressure is a relatively simple tool that has been used to
test the free radical theory.
D. Antioxidant Defenses
Cells are equipped with an impressive repertoire of
antioxidant enzymes, as well as small antioxidant mole-
cules mostly derived from dietary fruits and vegetables
(5, 351). These include 1) enzymatic scavengers such as
SOD, which hastens the dismutation of O20j to H2O2 , and
catalase and glutathione peroxidase (GPX), which convert
H2O2 to water; 2) hydrophilic radical scavengers such as
ascorbate, urate, and glutathione (GSH); 3) lipophilic radi-
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cal scavengers such as tocopherols, flavonoids, carot-
enoids, and ubiquinol; 4) enzymes involved in the reduc-
tion of oxidized forms of small molecular antioxidants
(GSH reductase, dehydroascorbate reductase) or respon-
sible for the maintenance of protein thiols (thioredoxin
reductase); and 5) the cellular machinery that maintains
a reducing environment (e.g., glucose-6-phosphate dehy-
drogenase, which regenerates NADPH). The complement
of defenses deployed differs not only between organisms
or tissues, but even between cellular compartments. For
instance, GPX plays an important role in mammals but is
absent from flies and nematodes (298, 330), and there
exist in humans three forms of SOD (cytosolic Cu,Zn-SOD,
mitochondrial Mn-SOD, and extracellular SOD), encoded
and regulated independently (91).
As far as supporting the free radical theory of aging
is concerned, the universality of antioxidant defenses is
good news. Although the nature of these defenses varies
between species, the presence of some type of antioxidant
defense is universal. In fact, some antioxidants, such as
SOD, are very highly conserved. Clearly, an indifference
to oxygen free radicals is inconsistent with life, underlin-
ing the centrality of oxidative damage. Moreover, the fact
that antioxidant defenses are not uniform has been incor-
porated into the free radical theory; differences in antioxi-
dant defenses between species have been put forth to
explain differences in life span. Although there is some-
thing uncomfortably ad hoc in these two different inter-
pretations of the data, they are not inconsistent. Whereas
aerobic life requires organisms to cope with oxidation to
some extent, different evolutionary pressures appear to
have selected for more or less investment in these de-
fenses, as is discussed in section III.
Finally, a persistent problem in testing the free radi-
cal theory is that antioxidants are both parallel (different
antioxidants can play similar roles, e.g., catalase and GPX)
and serial (enzymes operate in tandem to decompose radi-
cals to harmless products, e.g., SOD and catalase). Conse-
quently, measurements of individual antioxidant activities
do not have great relevance. In fact, as is discussed below,
pra APS-Phys Rev
554 KENNETH B. BECKMAN AND BRUCE N. AMES
measurements of age-related changes in individual antiox-
idants have led to conflicting results (297). For this reason,
aggregate assays have been devised, such as the suscepti-
bility of crude cellular homogenates to in vitro oxidation
by ionizing radiation (2, 300). Although these assays do
not provide any information about the specific mecha-
nisms of defense, they conveniently measure overall effec-
tiveness.
E. Repair of Oxidative Damage
Unlike defenses against oxidants, which have been
extensively characterized, the machinery for repairing ox-
idative damage is relatively unexplored. Nevertheless, it
is clear that cells repair oxidized lipids (e.g., phospholi-
pase A2 cleaves lipid peroxides from phospholipids; Ref.
239), oxidized nucleic acids (e.g., glycosylases specifically
recognize and excise oxidized bases from double-
stranded DNA; Refs. 23, 56a, 56b, 325), and oxidized pro-
teins (104, 240, 261, 316). The comparative biochemistry
of cellular repair is very fertile ground for the free radical
theory.
F. Synthesis: Interaction of Oxidant Generation,
Oxidative Damage, and Repair
The existence of multiple intracellular sources of oxi-
dants and complex defenses has led to refinements of
the free radical theory. For example, it is clear that the
metabolism of oxygen radicals is dynamic, with damage
resulting from an increase in oxidant generation or a de-
crease in antioxidant defenses. Consequently, a difference
in life span between species or individuals could be due
to different rates of living, or to different rates of scav-
enging (57, 82). The picture has been further complicated
by the discovery of specific enzymatic repair of oxidative
damage, leading to repair or fidelity versions of the
theory, in which life span is determined by the failure to
correct oxidative damage (137, 239).
The relationship among these three components of
oxidative stress: oxidant generation, antioxidant protec-
tion, and repair of oxidative damage, and the way in which
they have been investigated in testing the free radical
theory, is illustrated schematically in Figure 1. Increases
in oxidant generation, and decreases in antioxidant pro-
tection and repair systems, are among the theorys testa-
ble predictions and have been examined both as a func-
tion of age in individuals of the same species, as well as
between species of differing MLSP.
Finally, an extremely important (if experimentally re-
calcitrant) aspect of the interactions between oxidants,
antioxidants, and repair are feedback loops, positive and
negative, between them. Antioxidant defenses and cellu-
lar repair systems have been shown to be induced in re-
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Volume 78
sponse to oxidative challenges (67, 119, 320) and are of
course potential targets of oxidative destruction (145).
Also, the generation of oxidants may be enhanced by the
malfunctioning of oxidatively damaged molecules (28,
303). Therefore, with the examination of Figure 1, it is
not difficult to envision ways in which primary oxidative
destruction of any target (e.g., the components of the
mitochondrial ETC, scavenging enzymes such as SOD, or
DNA repair enzymes) might promote further oxidative
damage in what is frequently called a catastrophic vi-
cious cycle.
Although such cycles are intuitively appealing, their
documentation awaits future work and will be extremely
difficult from a technical standpoint. An alternative to lab-
based approaches, namely, the computational modeling
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of these complex interactions in what has been termed a
Network Theory of Ageing, is being pursued by theoreti-
cal gerontologists (170). Ultimately, a question of obvious
importance is whether or not such cycles, if they exist,
could be broken, and modeling may help pinpoint weak
links for therapeutic intervention.
III. REFINEMENTS AND COROLLARIES OF
THE FREE RADICAL THEORY
As the free radical theory has gained ground, it has
incorporated other ideas. For example, as mentioned
above, the rate of living hypothesis dovetailed with the
free radical theory once mitochondrial free radical genera-
tion was confirmed. Three other ideas that have been
influential are the evolutionary concept of antagonistic
pleiotropy, the somatic mutation theory of aging, and the
mitochondrial theory of aging.
A. Oxidants and Evolutionary Theories of Aging
The intracellular generation of oxidants capable of
limiting life span may appear paradoxical. It seems rea-
sonable to expect that natural selection might have de-
vised aerobic cells that do not leak toxic by-products.
Evolutionary biologists have contributed to the free radi-
cal theory by suggesting why physiologically harmful gen-
eration of oxygen radicals occurs. They have argued that
natural selection favors genes that act early in life and
increase reproduction, rather than genes that act to pre-
serve nongerm cells (the disposable soma), a principle
called antagonistic pleiotropy (162164, 265, 341).
The concept of antagonistic pleiotropy stresses that
in the wild, reproductive success is principally a function
of external factors. With the exception of modern-day
humans, individuals do not usually die of old age, but are
eaten, parasitized, or out-competed by others. Therefore,
preserving the cells of the disposable soma, otherwise
known as the body, may be disadvantageous if it detracts
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April 1998 FREE RADICAL THEORY OF AGING
from more pressing problems. The toxicity of respiratory
burst oxidants, for instance, cannot easily be eliminated
by evolution, since this would result in death from child-
hood infection. Similarly, an investment in improved anti-
oxidant defenses maximizes fitness only if the resources
are not better invested in strength, beauty, speed, or cun-
ning.
In terms of natural selection, the tremendous cost of
death before reproductive age, the constantly compound-
ing probability of death from external threats, and the cost
of failing to reproduce all ensure that selective pressure is
strongest at young ages. Any novel mutations that de-
crease oxidative damage first have to satisfy the criteria
of youthful reproduction. In short, selective pressure to
compete effectively at an early age may guarantee a cer-
tain degree of O2 toxicity and work against the conserva-
tion of the soma in the long run.
B. Oxidants and the Somatic Mutation Theory
of Aging
The somatic mutation theory holds that the accumu-
lation of DNA mutations is responsible for degenerative
senescence (23, 79, 212, 218, 332). In the case of cancer,
which results from both point mutations in oncogenes
and the loss of tumor suppressor gene function (often by
deletion), the role of mutations is unquestionable (5). It
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555
FIG. 1. The ultimate outcome of oxidative
stress is a function of 1) oxidant generation, 2)
antioxidant defenses, and 3) repair of oxidative
damage. Bolds arrows denote oxidative damage,
and dashed arrows denote routes for its preven-
tion or repair. Because of the ways in which
these processes may interact, multiple positive-
and negative-feedback loops are possible. Aging
(A) is situated at intersection of these processes.
In testing the free radical theory, changes in pro-
cesses 13 have been measured both as a func-
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tion of age and as a function of species maxi-
mum life span potential. The similarity of the
figure to the international emblem of radiation
is not a coincidence; the free radical theory has
its roots in radiation biology.
remains to be seen whether or not the argument is valid
for nonproliferative senescence. For instance, whereas
significant age-related increases in somatic mutations in a
reporter transgene (lacZ) have been measured in a mitotic
tissue of transgenic mice (the liver), no increase was de-
tected in the largely postmitotic brain of the same animals
(71a), suggesting that neurodegeneration, at least, is un-
likely to be the result of accumulated somatic mutations
in nuclear DNA. Moreover, the accumulation of mutations
in the liver tissues was not dramatic, suggesting that muta-
genesis may be of little functional consequence to mitotic
tissues as well (338b). In light of these data, what evidence
is there that somatic mutations are related to aging? A
compelling argument for the somatic mutation theory of
aging was provided years ago in the discovery that DNA
repair ability correlates with species-specific life span
(127a), a phenomenon that has recently been reconfirmed
(52a). However, it has been noted that DNA repair, which
is necessary for the prevention of tumorigenesis, is neces-
sary but not sufficient for longevity (52a). Ultimately, ar-
guments about the physiological significance of somatic
mutations hinge on how disruptive a given mutational
burden is to a cell or animal; with current methods, this
is an unanswerable question.
In any case, it has been demonstrated in numerous
studies with prokaryotes, yeast, and mammalian cells that
oxidants are mutagens, against which cells actively pro-
tect their genetic material (81, 108). Although it is not
pra APS-Phys Rev
556 KENNETH B. BECKMAN AND BRUCE N. AMES
yet clear what fraction of mutations can be attributed
to oxidative damage, the characterization and cloning of
defense genes against oxidative mutagenesis (17), and the
development of in vivo mutagenesis assays (198), has fi-
nally opened up avenues for definitive experiments.
C. Oxidants and Mitochondrial Theories of Aging
The mitochondrion has also long attracted attention
as one of the cells weak links, an organelle whose dys-
function has profound negative pleiotropic effects (193).
Mitochondria supply ATP and also sequester potentially
toxic Ca2/, yet because of their generation of O20j and
H2O2 , they are on the front lines of respiratory oxidative
stress. The idea that the mitochondrion is therefore
uniquely vulnerable was embraced early on by proponents
of the free radical theory (121). In the early 1980s, Miquel
and colleagues (84, 212, 213) proposed that oxidative dam-
age to mitochondrial DNA (mtDNA) in postmitotic cells
would lead to mutations and blocks to replication, and
consequently to mitochondrial dysfunction and physiolog-
ical decline. This mtDNA mutation hypothesis of aging,
which incorporates free radicals, somatic mutations, and
the central role of mitochondria in homeostasis, is pres-
ently under intense scrutiny (8, 11, 21, 50, 110, 222, 223,
238, 258260, 276, 288, 336, 339).
IV. OXIDATIVE PHENOMENOLOGY:
AGE-ASSOCIATED TRENDS
The phenomenological approach to the free radical
theory has involved looking for traces of oxidative dam-
age in vivo. Phenomenology is not well suited to critically
testing the free radical theory, since the data (which are
voluminous and generally supportive) mainly represent
correlations. Documented increases in oxidative damage,
no matter how impressive, may be a consequence of a
primary nonoxidative event. Nevertheless, phenomenol-
ogy is the foundation upon which more powerful experi-
ments depend, since the analytical methods developed for
it have been used to compare species, genetic mutants,
and populations with differing life spans. In fact, almost
all of the biomarkers of oxidative stress described in this
section have been found to accumulate at a faster rate in
short-lived species, and in many cases, this rate correlates
with O2 consumption. Familiarity with the most frequently
measured end points is a prerequisite to assessing the free
radical theory.
If oxidative damage is a significant cause of cellular
degeneration, then one expects to see more of it in older
individuals. Oxidative damage has been described in
terms of accumulation, modification, and depletion: ac- Inclusion of iron in the growth medium further increases
cumulation of end products of oxidative damage (such as
lipofuscin), modification of existing structures (such as
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Volume 78
oxidative adducts in DNA), and depletion (such as the
loss of enzymatic activity or reduced thiols).
A. Accumulation of Oxidative End Products
The gradual and steady accumulation of intracellular
yellow-brown fluorescent pigments, referred to as lipofus-
cin, occurs in numerous phyla. Lipofuscin arises promi-
nently in postmitotic cells (where, it is argued, it remains
undiluted by rounds of cell division; Ref. 296) and is lo-
cated in small granules in secondary lysosomes. Lipofus-
cin is structurally complex and variable, consisting mostly
of cross-linked lipid and protein residues (251, 296, 327),
and is ubiquitous, documented in species as diverse as
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nematodes, fruit flies, rats, bees, crab-eating monkeys, and
crayfish. Most important, it is abundant in aged tissues,
where it may occupy more than one-half of the volume
of the cell (347, 348).
Early on, it was discovered that incubation of amino
acids with the lipid peroxidation product malonaldehyde
under acidic conditions leads to the formation of lipofus-
cin-like fluorophores (42). The plausibility of such a reac-
tion, given the contents and low pH of lysosomes, sug-
gested that lipid peroxidation in vivo leads to the forma-
tion of lipofuscin (324). Other in vitro studies of lipid
peroxidation have since uncovered a great number of
routes to fluorescent, cross-linked products via promiscu-
ous oxidative chemistry, which suggests that lipofuscin is
a biomarker of lipid peroxidation (160, 296, 348).
Despite extensive in vitro experiments, it is not
known with certainty how lipofuscinogenesis occurs in
vivo, nor how lipofuscin comes to accumulate with age.
Lipid peroxidation could occur throughout the cell and
be followed by lysosomal phagocytosis and cross-linking
of peroxidative by-products, in which case an age-related
increase in lipofuscin content could be seen as the result
of oxidative damage. Alternatively, an age-associated de-
cline in lysosomal activity (due to something besides oxi-
dation) might increase the residence time of phagocy-
tosed material enough to enhance lipofuscinogenesis in
situ from constant amounts of peroxides (135). In support
of the latter possibility, infusion into rat brains of lyso-
somal proteinase inhibitors leads to the rapid accumula-
tion of lipofuscin-like granules (149). This scenario sug-
gests that lipofuscinogenesis may be a consequence, not
a cause, of aging.
Experiments with cultured cardiac myocytes have es-
tablished roles for both oxidative damage and lysosomal
turnover (28). Lipofuscin accumulates in these cells in
culture, and growth under increasing O2 partial pressure
from 5 to 40% markedly enhances its accumulation (306).
lipofuscinogenesis, and the iron chelator desferal de-
presses it, suggesting that Fenton reaction-generated jOH
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April 1998 FREE RADICAL THEORY OF AGING
is an initiator (200). Finally, antioxidants inhibit lipofuscin
formation in cultured cardiomyocytes, whereas lysosomal
protease inhibitors increase it (199, 201, 202).
Even if oxidative damage is primarily responsible for
depositing lipofuscin in the lysosomes of senescing ani-
mals, is it more than a biomarker of aging? It has been
theorized that lipofuscin accumulation is likely to impair
autophagy, as more lysosomal volume is occupied by the
indigestible material (28). Because lysosomes are respon-
sible for the recycling of materials and organelles, their
failure may include the following: 1) a delay in mitochon-
drial turnover (with a concomitant decrease in mitochon-
drial efficiency or an increase in mitochondrial oxidant
generation), 2) an accumulation of oxidatively modified
proteins and lipids in the cytosol awaiting degradation
(potentially aggravating cytosolic lipid peroxidation), 3)
an accumulation of lipofuscin-bound iron in a redox-ac-
tive form (which might promote further intralysosomal
lipid peroxidation), and 4) the disruption of lysosomal
membranes (and the spillage of hydrolytic enzymes into
the cytosol). Although these speculations (28) remain to
be substantiated, it has been shown that when treated
with sublethal doses of H2O2 , cultured cells display lyso-
somal disruption and leakage of the lysosomal compart-
ment into the cytosol (29). Also, it has been demonstrated
that the sensitivity of cultured primary hepatocytes to
oxidation, which was associated with a loss of GSH and an
influx of Ca2/, was prevented by the iron chelator desferal
(235). What is intriguing about these results is the fact
that whereas desferal stabilized the lysosomes, it did not
prevent the loss of GSH or the increase in intracellular
calcium, so it may be that it is lysosomal leakage per se,
rather than peroxidative damage, which is the actual le-
thal step in this model of oxidative killing.
B. Steady-State Levels of Oxidative Modification
Unlike cytosolic proteins whose half-lives are mea-
sured in minutes or hours, some extracellular proteins are
rarely recycled, and oxidative modification of these old
macromolecules occurs. A class of fluorescent cross-
linked molecules that is distinct from lipofuscin forms on
long-lived proteins such as collagen and lens crystallin
(216). These modifications are initiated by the reaction
of reducing sugars with free amino groups (glycation), a
chemical sequence that is unrelated to oxidation and re-
sults in a molecule known as an Amadori product. Further
nonoxidative rearrangements result in stable, cross-linked
advanced glycation end products (AGEs) (35), whose ab-
solute abundance appears to be an excellent biomarker
of age (217). Recently, it was discovered that oxidation
is one fate of the Amadori product. Pentosidine, the name
given to a cross-link involving arginine, lysine, and pen-
tose moieties, is one such glycoxidation product, the
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557
formation of which requires the presence of O2 (326). It
appears as if Amadori products themselves are a source
of H2O2 in vitro, which then accelerates glucose-mediated
fluorogenic collagen cross-linking in a catalase-sensitive
fashion, although it is unclear to what extent this occurs
in vivo (77, 217). As is the case with other AGEs, the
tissue burden of pentosidine is elevated in diabetics, as a
consequence of hyperglycemia.
Pentosidine has been found to accumulate as a func-
tion of age in shrews, rats, dogs, cows, pigs, monkeys,
and humans, yielding equivalently shaped curves in all
cases (284). It is not clear, however, how glycooxidative
modifications might contribute to degeneration. It has
been proposed that cross-linking in cartilage is related to
its decreased elasticity and relative resistance to proteoly-
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sis in old animals (9). However, the absolute amount of
collagen pentosidine cross-links attained at death is much
higher in long-lived than in short-lived species: 67 pmol/
mg in 3.5-yr-old shrews, 1518 pmol/mg in 25-yr-old mon-
keys, and 50100 pmol/mg in 90-yr-old humans (284). In
other words, it appears that the rate of pentosidine accu-
mulation may merely be a measure of more rapid oxida-
tive damage in short-lived species rather than an actual
cause of dysfunction.
An intriguing twist to this story has been the cloning
of a specific cellular receptor for AGE, called RAGE (re-
ceptor for AGE), that belongs to the immunoglobulin su-
perfamily and is expressed by mononuclear cells and the
vascular endothelium (277, 278). One of the effects of
AGE binding by RAGE is the generation (in mononuclear
cells) of intracellular oxidants, the activation of the oxi-
dant-sensitive transcription factor NFkB, and the induc-
tion of downstream events linked to atherogenesis (279,
280). As discussed in section XVB, it has recently been
shown that RAGE, which is highly expressed by microglial
cells in the brain (142), is a receptor for amyloid b-peptide
(Ab). The RAGE binding of Ab results in oxidant genera-
tion, implicated in the etiology of AD (293, 344).
Several amino acid residues in proteins are suscepti-
ble to oxidative modification, forming side chain carbonyl
derivatives (317). The development of sensitive methods
for the analysis of protein carbonyls by Stadtman and co-
workers (181) enabled them to study oxidative modifica-
tion in human brain tissue and cultured fibroblasts, and
in rat liver. They found a two- to threefold rise in protein
carbonyl content between young and old age, an increase
from 10 to 30% of the total protein pool (315). The in-
crease was exponential and correlated well with de-
creased activity of the oxidation-sensitive enzyme glu-
cose-6-phosphate dehydrogenase (G-6-PD). In compari-
son, the rise in protein oxidation in the mongolian gerbil
was less dramatic, increasingly significantly in brain,
heart, and testis, but not in kidney. As in human tissues,
trends for the activity of G-6-PD correspond to the in-
creased damage, falling in brain and heart but not in kid-
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558 KENNETH B. BECKMAN AND BRUCE N. AMES
ney (300). Similar results have been reported in an insect
model. An age-associated 2.5-fold increase in the protein
carbonyl content of old versus young houseflies has also
been documented (299), and as in humans, the increase
occurs exponentially during the life span. The similarity
of the degree and pattern of increase in insects and mam-
mals is striking, considering the enormous difference in
their MLSP (40 days vs. 100 yr). Moreover, protein car-
bonyl levels increase similarly in mitochondrial extracts
from the thoracic flight muscles of these animals (303).
Mitochondrial aconitase is particularly prone to oxidative
modification during aging in vivo and was identified by
the immunoblotting of housefly mitochondrial protein ex-
tracts with a monoclonal antibody designed to detect pro-
tein carbonyls (343b). Carbonylation of this key citric acid
cycle enzyme increased in parallel with a decline in its
activity.
Somewhat stronger evidence that protein oxidation
may play a causative role in senescence comes from com-
parisons of crawlers versus fliers of the same aging sults. Although some studies have reported a modest in-
cohort. Although the two groups share the same chrono-
logical age, crawlers are phenotypically senescent individ-
uals that have lost the ability to fly and have a shorter
remaining average life span than do fliers (e.g., 9.0 days
vs. 13.3 days for 10-day-old crawlers and fliers, respec-
tively). The protein carbonyl content of crawlers was 29%
higher than that of fliers (299), reflecting their greater
phenotypic age, as was the degree of carbonyl modifica-
tion of mitochondrial (but not cytosolic) aconitase (343b).
Humans suffering from Werners syndrome, a disease
characterized by premature senescence, are individuals
whose phenotypic aging is also accelerated, and they too
appear to have more extensive protein oxidation. Fibro-
blasts from Werners patients of all ages have a level of
protein carbonyls equivalent to that in 80-yr-old controls
(233). In a creative study attempting to correlate protein
oxidation to a physiologically relevant end point, it was
shown that in old mice, interanimal variation in protein
carbonyl content of two different areas of the brain (cere-
bral cortex vs. cerebellum) was associated with parallel
interanimal variation in memory and motor function defi-
cits (90).
Are protein carbonyls physiologically relevant, or are
they merely markers? What are the actual consequences
of protein modification? Unfortunately, there are few
quantitative data with which to answer this question, al-
though qualitative data exist. The fate of oxidized proteins
may depend on the form of damage. For example, metal-
catalyzed oxidation of G-6-PD by iron/citrate results in a
thermolabile enzyme that is a better substrate for proteol-
ysis than is the native enzyme (93). Rapid turnover of
metal-oxidized G-6-PD may therefore proceed efficiently.
On the other hand, G-6-PD modification by 4-hydroxy-2-
nonenal, a lipid peroxidation product, also inactivates the
enzyme but does not render the enzyme thermolabile or
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Volume 78
increase its degradation by proteases (322). This differ-
ence exists despite the fact that in both cases, the same
lysine residue is affected. To make matters more complex,
the cross-linking of G-6-PD multimers by 4-hydroxy-2-non-
enal (which predictably results in a product with lipofus-
cin-like fluorescence) produces a molecular species that
actually inhibits the multicatalytic protease (92). The
physiological cost of protein oxidation is presently an un-
known quantity.
The appearance of protein-bound 3,4-dihydroxyphe-
nylalanine (DOPA) on jOH-damaged proteins has been
characterized; when converted to a quinone, protein-
bound DOPA can undergo redox cycling, generating
O20j. It has therefore been proposed that protein oxidation
may contribute to the progression of aging not merely by
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the loss of protein function, but also by an acceleration
of the flux of oxidants (61, 63, 64, 69, 101, 102).
The oxidative modification of DNA has also been
studied in animals of different ages, with conflicting re-
crease in specific oxidative adducts, single-strand breaks,
and abasic sites, others have been negative (23, 132, 156,
221, 337). The failure to detect an age-related increase
in oxidative adducts by the analytical chromatographic
techniques typically employed may have been due to the
difficulty of working close to the limit of sensitivity (17).
In fact, it has become apparent that the measurement of
the adduct oxo8dG is frequently plagued by artifacts (29a,
44b, 127c, 156a, 248a) and that these may have compro-
mised some published experiments. Of particular concern
are measurements of oxo8dG in mtDNA (16), which have
generally been higher than in nuclear DNA, but which
may be particularly prone to artifacts associated with the
analysis of small samples (16, 127c). Moreover, it is note-
worthy that even among the highly variable published esti-
mates of oxo8dG in mtDNA are values that are equivalent
to the lowest measured values of oxo8dG in nuclear DNA
(131a). Because of the small number of studies of mtDNA
and the high variability between the measured values, it
is not yet possible to conclude that mtDNA is, in fact,
more heavily oxidized than nDNA. Encouragingly, alterna-
tive PCR-based methods for measuring oxidative damage
have recently been used to compare oxidation of mtDNA
and nDNA by exogenous oxidants, with the result that
the former appears more sensitive than the latter (270a,
343a), although these studies could not quantify baseline
values of damage. With methodological improvements, fu-
ture experiments may be more conclusive. For instance,
the use of single-cell gel electrophoresis (the comet assay)
to measure single-strand breaks and abasic sites in whole
rat hepatocytes in situ revealed a statistically significant
1.5-fold increase in old rats compared with young rats
(131) (although this experiment did not distinguish be-
tween oxidative and nonoxidative damage).
In any case, even if the burden of oxidative adducts
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April 1998 FREE RADICAL THEORY OF AGING
does increase with age, there is virtually no information
about the likely effect of oxidative DNA damage in vivo,
apart from the knowledge that it leads to mutations and
cancer. The fact that there is active DNA repair in postmi-
totic tissues (in which the danger of mutation due to repli-
cation is nonexistent), and that such repair is often tar-
geted to transcribed regions of the genome, suggests that
DNA damage itself interferes with gene expression and is
not tolerated (116, 117). This important question deserves
more attention.
C. Oxidative Depletion of Biochemical Pools
The oxidative depletion of molecules with increasing
age has not been well documented in senescent animals,
since the destruction of molecules does not often leave
traces; luckily, some pathways of oxidative damage do
leave biochemical fingerprints. The loss of integrity of
lipid bilayers due to peroxidation is one of the most salient
effects of oxidative damage (324) and results in the gener-
ation of aldehydes and alkanes. Unfortunately, these are
not easily measured, the widespread use of the simple
and nonspecific thiobarbituric acid test notwithstanding
(109). Nevertheless, countless studies have reported an
increase in thiobarbituric acid-reactive substances
(TBARS) with age. Combined with other more reliable
assays, these studies have demonstrated that there is a
greater degree of lipid peroxidation in older animals (209).
The measurements of exhaled ethane and pentane is a
technique that has the advantage of being applicable to
humans (165). Unlike lipofuscin and TBARS, which mea-
sure the size of a pool of destroyed molecules and require
a tissue biopsy, the assay of exhaled hydrocarbons mea-
sures the rate of damage and is noninvasive. Breath pen-
tane has been found to increase significantly with age in
humans, suggesting that increased lipid turnover occurs
with age because of peroxidation (161a, 208, 356). Re-
finement of the technique and elimination of the associ-
ated artifacts (314) should facilitate further testing of the
free radical theory in humans.
The loss of activity of several oxygen-sensitive en-
zymes (G-6-PD, glutamate synthetase) has been reported
in mammalian models of aging (315). In houseflies, a de-
cline in G-6-PD, glutamate synthetase, and alcohol dehy-
drogenase activities has also been documented and coin-
cides with a dramatic loss of protein sulfhydryls (3). An-
other commonly reported age-related loss is an increase
in the ratio of oxidized to reduced glutathione, which may
reflect a disruption of the cells redox state (215, 273).
D. Age-Associated Trends in Antioxidant Defenses
and Repair
What is the cause of age-related oxidative damage?
It could result from less active antioxidant defenses and
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559
repair, but studies that have measured age-related
changes in antioxidant defenses have generated conflict-
ing results. Recent measurements of antioxidants in mon-
golian gerbils (300) and mice (215) are representative of
the types of patterns that have been uncovered in many
other studies (65, 248, 254, 263, 274, 301, 302, 305, 329).
In various tissues of gerbils, there was not a consistent
pattern of change; increases in SOD and decreases in GSH
were observed, whereas GPX was equivalent at different
ages and catalase increased or decreased, depending on
the tissues and the age at analysis. In mouse brain, on the
other hand, significant decreases in SOD, catalase, and
GSH reductase were observed, although GPX levels were
unchanged.
Another complication is that defenses are induced in
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response to stress. Therefore, a higher level may indicate
better protection, or alternatively, greater need for antiox-
idant defenses due to an increase in oxidant generation.
Studies of antioxidants in rat heart and skeletal muscles
illustrate this point. In heart, decreases in cytosolic SOD
and GPX and increases in mitochondrial SOD and GPX
were noted in older animals, and several indexes of oxida-
tive damage were also elevated (151). From these results,
it was concluded that although overall myocardial antioxi-
dant defenses were weakened in the older animals, they
were induced in mitochondria as a compensatory re-
sponse. In skeletal muscles, in contrast, increases were
observed in both cytosolic and mitochondrial forms of all
of the enzymes studied (150), despite the fact that indexes
of lipid peroxidation were again elevated; in this case,
it was concluded that both cytosolic and mitochondrial
antioxidants were induced. The credibility of these
hypotheses is not in question, but it is hard to see how
they could be disproved. When these and similar studies
of age-related antioxidant levels are combined, what re-
mains is a confusing assemblage of ambiguous trends.
Of course, interactions between antioxidants are
complex, which aggravates the problem. To avoid the
problems posed by assays of individual antioxidants, ag-
gregate measures of antioxidant defenses have been de-
vised. A crude but integrative measure of antioxidant de-
fenses, for instance, is the susceptibility of a homogenate
to induced oxidation. X-irradiation of a whole body ho-
mogenate of houseflies results in a linear, dose-dependent
increase in protein carbonyls. When homogenates of old
and young flies are compared, the rate of induction of
protein carbonyls by X-irradiation is 45% higher in 14-
than 5-day-old flies. This suggests that the antioxidant
defenses in older flies are less able to cope with oxidative
stress. Moreover, the activity of G-6-PD, an enzyme known
to be sensitive to oxidation, decreases upon X-irradiation
of living flies, and does so to a greater extent in old than
young animals (2). When this assay was applied to the
gerbil samples described above, in which no overall
change in antioxidants was seen, a clear difference be-
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560 KENNETH B. BECKMAN AND BRUCE N. AMES
tween young and old tissues emerged. Whereas 6 krad of
X-irradiation induced a 2038% increase in protein car-
bonyls in 5-mo-old animals, it induced a 152211% in-
crease in 26-mo-old animals (300). Similarly, although syn-
aptosomes from young and old mice contain equivalent
amounts of ATP and GSH, those of old mice were far
more sensitive to GSH depletion by the diethyl maleate
than those from young mice (197). Lastly, reperfusion in-
jury is a well-established model of oxidative stress associ-
ated with the reestablishment of blood flow following
ischemia, and it causes greater oxidative damage to heart
tissues of old rats than young ones (192a). The use of a
polyclonal antiserum specific for adducts between lipid
peroxidation end products and proteins detected such co-
valent modifications of mitochondrial proteins from old
but not young animals, which was associated with a more
dramatic loss of respiratory capacity in the former.
Whereas the baseline mitochondrial respiratory parame-
ters (before ischemia-reperfusion) did not differ between
young and old animals, the administration of a physiologi-
cally relevant stress revealed a probable age-related de-
cline in antioxidant defenses.
Another alternative to measuring absolute levels of
antioxidants in old versus young animals is to investigate
the ability of animals of different ages to induce antioxi-
dants, an approach that has been applied to the analysis
of SOD in the nematode Caenorhabditis elegans (59).
Whereas in young animals challenge with hyperoxia or
the redox cycling compound plumbagin resulted in an
increase in SOD activity, in middle-aged or old animals it
actually resulted in a net loss of activity.
What about repair of oxidative damage? Does its ac-
tivity decrease with age? The bulk of evidence suggests
that there is probably not an overall age-associated
change in the intrinsic ability of cells to degrade damaged
proteins (94, 270). Although a dramatic decrease in the
activity of the oxidized protein-specific alkaline protease
has been reported in old rat hepatocytes (318), no change
in this activity was measured in the heart or brain of 25-
versus 5-mo-old gerbils (300). In a separate work, a 50%
age-related decline in a single activity (peptidylglutamyl-
peptide hydrolase activity) of the hepatic multicatalytic
protease was associated with its selective sensitivity (rela-
tive to the multicatalytic proteases other activities) to
metal-catalyzed oxidation, suggesting that resistance to
oxidants may (logically) characterize the proteases re-
sponsible for degrading damaged proteins (46).
Although the intrinsic protease activity may not de-
crease with age, there is evidence that repair of oxidized
proteins may be less easily induced in response to an
oxidative insult in old animals. For instance, exposure of
young and old rats to 100% O2 increased the content of
protein carbonyls in both groups over a 48-h period. Be-
tween 48 and 54 h of exposure, however, alkaline protease
activity was induced in young animals, with a correspond-
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Volume 78
ing decrease in protein carbonyls to initial levels. In old
animals, on the other hand, no increase in activity was
observed, and protein carbonyl levels continued to rise
throughout the time course (318).
There is circumstantial evidence from mutagenesis
studies that either antioxidant defenses or repair of oxida-
tive DNA damage (or both) is less efficient in old mice.
The induction of somatic mutations in mice by g-irradia-
tion is from 2.3- to 3.6-fold higher in old than in young
animals, depending on the dose (99). The induction of
mutations in young and old animals was reduced by feed-
ing the animals a cocktail of dietary antioxidants, con-
firming that oxidants played a mutagenic role in these
experiments. Therefore, the more pronounced induction
of mutations in older mice is indirect evidence of de-
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creased antioxidant defenses and repair (99). Later exper-
iments employing peripheral lymphocytes from young and
old human subjects resulted in similar results (100). The
ability of human peripheral lymphocytes to repair oxida-
tive DNA damage induced by H2O2 has also been found
to be less efficient in cells from older donors (14).
Altogether, the results above suggest that older cells
may be less able to prevent oxidative damage from oc-
curring, and less effective at removing the damage once
it has occurred. There is a clear need for more and better
data about age-related trends in defenses and repair.
E. Age-Associated Trends in Oxidant Generation
The accumulation of oxidative damage could also re-
sult from an age-associated increase in the primary gener-
ation of oxidants, and some research suggests that this is
the case. Generation of H2O2 and O2j0 by isolated mito-
chondria and submitochondrial particles from 25-mo-old
gerbils, for instance, is 150200% that of 5-mo-old ani-
mals (300), and that of aged rat heart (305) and brain
(93a) has also been reported to be elevated. On the other
hand, a recent study that paid specific attention to main-
taining physiological substrate concentrations during in
vitro mitochondrial incubations failed to detect an age-
related increase in mitochondria H2O2 output (118). Re-
cent measurements of oxidant generation in carefully
isolated rat hepatocytes from young and old animals, em-
ploying an intracellular dye that fluoresces upon oxida-
tion, have confirmed under conditions that preserve cellu-
lar integrity that cellular oxidant generation appears to
increase (110). A similar increase in oxidant generation
documented in flight muscle mitochondria of houseflies
was associated with increases in the activities of every
measured component of the electron transport chain ex-
cept for the content of UQ (308), suggesting that an imbal-
ance in electron transport may be the cause of aberrant
reduction of oxygen. In another experiment, a population
of chronologically identical 12-day-old flies was separated
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April 1998 FREE RADICAL THEORY OF AGING
into phenotypically older crawlers and younger fliers as
described above; mitochondrial H2O2 generation was
twice as high in the crawlers than in the fliers, reflecting
their greater phenotypic age.
Interestingly, oxidative damage to mitochondrial
membranes and proteins has itself been implicated in en-
hanced oxidant generation; exposure of isolated mito-
chondria to the free radical generator 2,2-azobis(2-amino-
propane)dihydrochloride or the cross-linking agent glutar-
aldehyde resulted in mitochondria that were more able to
generate H2O2 when fed exogenous substrate (308). When
these in vitro studies are combined with the above evi-
dence of increased oxidative damage, the picture that
emerges is a potential vicious cycle of oxidative damage
and oxidant generation.
V. INTERSPECIES COMPARISONS
All of the data discussed in the previous section iden-
tify age-related differences within a given species. A com-
plementary, and in some ways more powerful approach,
is to compare species that have different MLSP. Compara-
tive biochemistry and physiology, inspired by the rate of
living hypothesis, has played a key role in establishing
the free radical theory. Several representative studies are
described below.
A. Oxidative Damage and Maximum
Life Span Potential
The accumulation of cardiac lipofuscin in the monkey
correlates with its cumulative O2 consumption, starting at
sexual maturation (226). Comparisons between rodent,
dog, and primate species also revealed a close correlation
between specific metabolic rate and lipofuscin accumula-
tion (224, 225). The accumulation of the glycooxidation
product pentosidine is similar. Short-lived species such
as the shrew (MLSP 3.5 yr) or rat (MLSP 3 yr), with
high specific metabolic rates, accumulate pentosidine in
collagen at a rate of 0.30.5 pmol/mg annually. Longer-
lived primates such as monkeys and humans accumulate
pentosidine at about one-tenth as fast (284).
Interspecies comparisons of protein oxidation mirror
these results. The protein carbonyl content of 15-day-old
individuals of five species of fly correlated negatively with
mean life span; the longest-lived species (Drosophila mel-
anogaster, mean life span 65.5 days) had roughly one-
third the level of protein oxidation as shortest-lived spe-
cies (Phaenecia sericata, mean life span 29.5 days)
(310). The content of protein carbonyls was 20% higher
in the heart, and 50% higher in the brain, of 3.5-mo-old
individuals of Mus musculus (MLSP 3.5 yr) than in 3.5-
mo-old individuals of Peromyscus leucopus (MLSP 8
yr) (304).
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561
The excretion of the oxo8gua and oxo8dG in urine
is a reflection of whole body oxidative hits to DNA (287).
The validity of its use to measure in vivo oxidative hits is
strengthened by recent study of 33 women, in which both
O2 consumption and excretion of oxo8dG were measured.
There was a highly significant positive correlation (P
0.00007, r 0.64) between O2 consumption and excretion
of adducts (188). When the urinary output of the oxidative
DNA repair products oxo8gua thymine glycol and thymi-
dine glycol were compared in mice, rats, and humans,
they were found to correlate with species-specific meta-
bolic rate (1, 287).
B. Antioxidant Defenses and Maximum
Life Span Potential
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One version of the free radical theory proposes that
the differences in life span between species are due to
species-specific antioxidant capacity. Early work by Cut-
ler (57) tested this association: MLSP correlated positively
with SOD but negatively with catalase and GPX (57). Re-
cently, other groups have revisited this hypothesis, with
similar results. Comparisons between SOD, catalase, GPX,
and GSH in brain, liver, and heart from mice, rats, guinea
pigs, rabbits, pigs, and cows were carried out and revealed
that 1) SOD and catalase activities correlated positively
with MLSP, 2) GSH activity correlated negatively with
MLSP, and 3) GPX correlated positively with MLSP in the
brain and negatively in the liver and heart (309).
Interspecies comparisons between more distantly re-
lated vertebrate classes found either no correlation or a
negative correlation between antioxidants and MLSP. For
instance, in the liver of fish, frogs, birds, and mammals,
strongly significant negative correlations were found for
catalase, GPX, and GSH, whereas all other measured anti-
oxidants (SOD, GSH reductase, ascorbate, urate, GSH)
failed to correlate with MLSP (190). When the same anti-
oxidants were measured in brain and lung tissues of the
same species, virtually identical results were obtained
(12, 247).
Comparisons between two very closely related spe-
cies of mice, the house mouse (M. musculus) and the
white-footed mouse (P. leucopus), which lives twice as
long, have revealed higher levels of SOD, catalase, and
GPX in brain and heart extracts of the latter, with the
differences in GPX being the most dramatic. Also, when
brain homogenates of the two species were challenged
with X-irradiation, protein carbonyls accumulated at a
threefold higher rate in shorter-lived M. musculus than
longer-lived P. leucopus (304).
Together, these results suggest that the evolution of
longevity has not been associated with a clear-cut in-
crease in antioxidant capacity, at least across the broad
sweep of evolution. (The comparisons between M. mus-
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562 KENNETH B. BECKMAN AND BRUCE N. AMES
culus and P. leucopus invite the speculation that the more
recent radiation of long-lived species may be associated
with the coordinate upregulation of antioxidant defenses.)
In any case, what emerged from the comparative biochem-
istry of free radical defenses is a fascinating paradox:
birds, which typically have much longer long life spans
than rodents, have lower activities of antioxidants and
higher metabolic rates. This paradox has inspired the in-
terspecies comparisons of mitochondrial oxidant genera-
tion discussed in section V.
C. Generation of Reactive Oxygen Species and
Maximum Life Span Potential
The rate of living hypothesis identified an inverse
correlation between metabolic rate and life span, and the
free radical theory supplied a convenient mechanistic
link: the mitochondrial generation of oxidants. Appealing
as this theory is, one of the problems with the rate of
living hypothesis is the conspicuous lack of fit of a few
groups, notably birds and primates. Both of these groups
live longer than their specific metabolic rate would pre-
dict. As a result, the total lifetime oxygen consumption of
these groups is quite a bit higher than other groups (e.g.,
during their MLSP, pigeons and canaries consume 465 and
1,222 liters O2/g, respectively, whereas mice, rats, guinea
pigs, and trout consume 77, 28, 48, and 26 liters O2 , respec-
tively) (247, 248). However, it cannot be assumed that the
generation of oxidants is a direct function of metabolic
rate, since mitochondria of different species (or even tis-
sues) may exhibit different rates of intrinsic oxidant gen-
eration. These lines of reasoning by two laboratories have
led to a similar line of experimentation, the results of
which suggest that longevity is associated with a lesser
capacity for mitochondrial oxidant generation (13, 172,
173, 304, 310).
For example, isolated pigeon mitochondria from
brain, liver, or heart consume from two- to threefold as
much oxygen as isolated rat mitochondria from the same
tissues. However, pigeon mitochondria generate only one-
third to one-half as much H2O2 as rat mitochondria under
the same conditions. As a result, the calculated percent-
age of O2 converted to O20j/H2O2 by mitochondria from
lung, liver, and brain is 10-fold lower in pigeons than in
rats. This lower rate of oxidant generation corresponds
with the roughly 10-fold longer life span of pigeons (13).
A second, independent comparison of mitochondrial oxi-
dant generation by pigeons and rats found similar results
(172). Detailed respiratory comparisons have shown that
generation of oxidants from both complex I and complex
III of the mitochondrial electron transport chain is lower
in pigeon than rat (130a).
In fact, the earliest interspecies study of mitochon-
drial oxidant production compared liver mitochondria of
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Volume 78
five mammalian species and thoracic muscle mitochon-
dria of two species of fly (311), and also found that longer
MLSPs were associated with lower mitochondrial oxidant
generation. The mitochondria from flies produced from
6-fold more to 300-fold more oxidants than those from
mammals. In a more recent comparison of heart tissues
of eight diverse mammalian species of widely varying life
span, submitochondrial particles of the short-lived species
were found to generate more O20j than those of long-lived
species, a property correlated directly with the concentra-
tion of CoQ9 (coenzyme Q possessing 9 isoprene units in
its isoprenoid tail) and inversely with CoQ10 (although
experiments intended to demonstrate a direct relationship
between CoQ9:CoQ10 ratio and O20j generation failed to do
so) (178a).
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Interestingly, it appears as if the concept of intrinsic
mitochondrial radical generation may also partially ex-
plain longevity differences between more closely related
species. Measurements of O20j and H2O2 generation by
isolated mitochondria from the mouse species M. muscu-
lus and P. leucopus revealed that the former, which live
half as long as the latter, also generate 4874% more
O20j and 300500% more H2O2 . [As is the case with rats
and pigeons, long-lived P. leucopus has the higher specific
metabolic rate of the two species (304).] Finally, a com-
parison of five species of dipteran fly whose mean life
spans vary by twofold also showed that the rate of genera-
tion of mitochondrial O20j and H2O2 correlated negatively
with life span (310).
Together, interspecies comparisons of oxidative dam-
age, antioxidant defenses, and oxidant generation provide
some of the most compelling evidence that oxidants are
one of the most significant determinants of life span.
VI. DIETARY RESTRICTION
Limiting the dietary intake of mammals is a well-
established way to extend life span (340). Interestingly,
early expectations that dietary restriction (DR) would
lower metabolic rate per se have not been confirmed, and
so if DR attenuates oxidative damage, it is not via a simple
reduction in oxygen consumption (312, 339a). Rather,
there is now convincing evidence that dietary restriction
may act in part by decreasing oxidative stress and increas-
ing antioxidant defenses and repair (352). For example,
mice whose caloric intake was restricted by 40% (DR)
compared with ad libitum (AL) fed controls, and who
lived 43% longer on average, had a less rapid accumulation
of protein carbonyls in brain, heart, and kidney. Moreover,
the generation of H2O2 and O20j by mitochondria and sub-
mitochondrial particles from DR animals was lower than
from AL animals, and also increased less with advancing
age. In this study, however, DR did not appear to enhance
antioxidant defenses (305). In another study, 40% DR of
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April 1998 FREE RADICAL THEORY OF AGING
rats increased the activities of SOD, catalase, and GPX at
older ages; free radical damage, as measured by TBARS
and lipofuscin accumulation, was correspondingly lower
(253). In a third study, focusing on mitochondrial oxidant
generation and membrane properties of synaptosomal
preparations, 40% DR reduced mitochondrial oxidant gen-
eration in both young and old samples and prevented the
age-dependent decline in membrane fluidity, despite the
fact that increases in the cholesterol-to-phospholipid ratio
were common to both groups of animals (43). Other ex-
periments have been recently reviewed (335, 352).
Dietary restriction also delays cancer incidence,
which may be a reflection of fewer mutations induced by
oxidants in DR animals. Dietary restriction has been
shown to strengthen DNA repair (111), although few stud-
ies have focused specifically on repair of oxidative lesions.
Not only caloric restriction, but also restriction of protein
intake, can forestall the occurrence of cancer in rodent
models. Although the mechanism by which protein re-
striction operates is not clear, enhanced protection
against cellular degeneration, including oxidative damage,
is a likely candidate. It is therefore interesting that the
level of protein carbonyls in animals fed a low-protein
diet was significantly lower than in animals fed standard
lab food and that treatment of the animals with g-irradia-
tion induced a greater increase in protein carbonyls in
high- than in low-protein animals (350).
VII. MANIPULATION OF RATE OF LIVING
The metabolic activity of caged houseflies can be re-
duced by limiting the space available for flight (low activ-
ity LA vs. high activity HA). It is expected that the
LA flies should live longer due to lower O2 consumption
and generation of oxidants, and this is in fact observed.
LA flies exhibit more than a twofold increase in both mean
life span and MLSP. Measurements of protein carbonyls
in 14-day-old LA and HA flies, which have not yet begun
to die, revealed an elevation of more than 50% in HA flies
relative to LA flies in both whole body extracts (299) and
in mitochondria (303).
Under unfavorable conditions, the nematode C. eleg-
ans may undergo a morphological transformation to form
a dauer larva, a resting stage that does not feed, exhibits
altered metabolic activity, and can survive for several
times the MLSP of adult nematodes before returning to
the adult stage. Interestingly, dauer larvae have also been
found to have increased SOD activity (178).
VIII. MANIPULATION OF OXYGEN TENSION
Another experimental manipulation that modulates
life span is growth under different O2 tensions. The growth
of houseflies in an atmosphere of 100% O2 , for example,
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563
markedly reduces their mean and MLSPs, and also in-
creases the rate of accumulation of protein carbonyls in
whole body extracts (299) and in isolated mitochondria
(303). Similarly, elevated atmospheric O2 decreased, and
subnormal oxygen increased, the mean and maximum life
spans of nematodes (138). Of course, the principal draw-
backs of this type of experiment are its inapplicability
to mammals and the fact that elevations or decreases in
ambient O2 in such species will likely be confounded by
the overt pathology of hypo- or hyperoxia.
IX. SUPPLEMENTATION WITH DIETARY
ANTIOXIDANTS
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As one of the most intuitive approaches to testing
the free radical theory, nutritional supplementation has
been attempted with numerous species and compounds,
with the result that mean life span has been extended
in some instances (122). In mammals, results have been
mixed. To cite recent studies, life-long oral supplementa-
tion of rats with UQ was without effect on mortality (189),
whereas supplementation of the senescence accelerated
mouse strain (see sect. XII) with bCATECHIN, a com-
mercial supplement with antioxidant activities (174, 349)
resulted in life extension (175). Negative results from the
feeding of antioxidants have been rationalized by arguing
that many of the antioxidants fed to mammals interfere
with mitochondrial respiration, and so the failure of anti-
oxidant trials to extend MLSP may have been due to their
toxicity (127). In a recent short-term feeding experiment,
in contrast, the thiol donor N-acetyl-cysteine was found
to markedly improve mitochondrial ETC complex activity
in rats (214) and short-term feeding with a complex anti-
oxidant-containing plant extract attenuated oxidative
damage in rat mitochondria (272a). Experiments aimed
at extending life span should probably be preceded by
such short-term trials, to document the uptake, distribu-
tion, and biochemical action of the compounds.
Although no large-scale human nutritional interven-
tion trials have been aimed specifically at the study of
aging, a few have been undertaken in the hopes that di-
etary antioxidants might help prevent cancer. Three simi-
lar and heavily scrutinized studies are the a-tocopherol
b-carotene (ATBC) prevention study (4), the physicians
health study (129), and the b-carotene and retinol efficacy
trial (CARET) (236), all of which were designed to assess
the effect of antioxidants on lung cancer risks. Although
the details of these enormous experiments are not im-
portant to this discussion, the overall result was instruc-
tive: there was either no decrease or a modest increase
in lung incidence and mortality with b-carotene adminis-
tration.
These negative results, as well as negative results
described above using laboratory rodents, should not be
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564 KENNETH B. BECKMAN AND BRUCE N. AMES
taken as evidence that the free radical theory of aging is
flawed. In fact, they prove merely that a complex organism
like a human or rodent is unlikely to respond predictably
to crude manipulations such as supplementation with one
or a small number of compounds (22), as well as that a
single end point (such as lung cancer) is not equivalent
to aging. Since the time when the results of these human
trials were announced, a number of explanations have
been forwarded to explain the paradoxical promotion of
cancer by b-carotene (252), which indicates that the ulti-
mate value of these trials may be a more precise under-
standing of this antioxidant. Ultimately, what the entire
experience should teach the field of molecular gerontol-
ogy is that at least until the biochemistry of dietary and
cellular antioxidants is better understood, dietary trials in
laboratory animals or humans will remain unreliable tests
of the free radical theory; molecular gerontology should
focus on more instructive experiments.
X. ADMINISTRATION OF PHARMACOLOGICAL
ANTIOXIDANTS
Dramatic antiaging effects have been observed by
Floyd and co-workers (234) with the free radical spin trap
N-tert-butyl-a-phenylnitrone (PBN). Initial studies used
PBN as a spin trap to measure radical generation during
ischemia-reperfusion injury of gerbil brain (234). During
reperfusion, the appearance of protein carbonyls corre-
lated well with the loss of glutamine synthetase activity
and the appearance of PBN-dependent nitroxide radical.
Interestingly, it was also discovered that pretreatment
with PBN attenuated protein oxidation and loss of gluta-
mine synthetase activity and reduced lethality.
In a subsequent study that investigated the effect of
PBN on spontaneous protein oxidation, it was discovered
that twice-daily administration of PBN to old gerbils (32
mg/kg) reversed age-related oxidative damage (34). For
example, during normal aging of gerbils, the level of brain
protein carbonyls increased 185%, and glutamine synthe-
tase activity declined by 35%. A marked drop in neutral
protease activity was also observed. Old animals adminis-
tered PBN for 2 wk, however, experienced a reversal of
these trends: protein carbonyls dropped, and glutamine
synthetase and neutral protease activities were virtually
restored. This effect was reversible, since over a 2-wk
period after cessation of PBN treatment, the oxidation of
proteins increased, and the activities of glutamine synthe-
tase and neutral protease fell. Most impressive, however,
was the performance of the PBN-treated old gerbils in a
test of short-term memory; whereas the old gerbils were
twice as likely to make errors as young gerbils, the old
animals treated with PBN performed as well as the
young (34).
Although these preliminary results held great prom-
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ise, not only for the study of aging, but also for its thera-
peutic treatment, subsequent experiments reported that
the results were irreproducible (31, 32). Further follow-
up work from a third laboratory confirmed the initial find-
ings in gerbil brain, but at the same time found no effect
of PBN in lowering the level of protein carbonyls in gerbil
heart or mouse brain (75). The chronic treatment of aged
rats with PBN was found to reverse age-related cognitive
impairment (294). Finally, the administration of PBN from
age 20 wk throughout the life of a short-lived strain of
mouse [the senescence accelerated mouse (SAM)] in-
creased its life span from 42 to 56 wk (an increase of
33%) (73). As is argued (in sect. IX) to be the case for
experiments with dietary intervention, maximum worth
will be derived from pharmacological experiments when
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they include not only end-point mortality measurements
but more focused studies of cellular effects.
XI. IN VITRO SENESCENCE AND OXIDANTS
The growth of normal diploid fibroblast in vitro is
limited; after a specific number of population doublings
(PDs) characteristic of the species, tissue, and age of the
donor (the Hayflick limit, named for the phenomenons
discoverer), the cells cease replication, which culminates
in a viable, nondividing population with characteristic se-
nescent morphology (56). Research on such replicative
senescence, and the vigorous debate about its relevance
to organismal aging, have been recently reviewed (30, 55,
56). For the purposes of this discussion, a few points
are worth reiterating. 1) There are numerous correlations
between organismal MLSP and replicative senescence. Fi-
broblasts from species with shorter MLSP exhibit a
shorter in vitro life span than cells from more long-lived
species. Similarly, fibroblasts from older individuals pos-
sess a shorter in vitro replicative potential than those from
younger individuals (30), although this generalization has
been questioned (55). Moreover, cells from patients with
Werners syndrome, a disease of accelerated aging (see
sect. XVII), also complete fewer PDs before crisis, when
replication ceases (30). 2) In vitro senescence may be
relevant to organismal life span in at least two ways. First,
as one of the likely components of tumor suppression,
cellular senescence is necessary (but not sufficient) for
extended life span. In other words, a long life span (as in
humans, for instance) requires the absence of cancer at
younger ages, and this is achieved in part by growth arrest
of preneoplastic cells by mechanisms that operate during
in vitro senescence (30, 269). Support for this assertion
are the observations that fibroblasts from rodents (which
have a significant cancer incidence after 23 yr of age)
spontaneously immortalize in vitro, whereas fibroblasts
from humans (who are relatively free of cancer until much
later ages) do not (30), and that immortalization is associ-
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April 1998 FREE RADICAL THEORY OF AGING
ated with inactivation of p53 and Rb, which are frequently
mutated in neoplasms (107). Second, senescent cells ap-
pear to occur in vivo, as judged by enzymatic markers
characterized in vitro (68). It has been suggested that even
at a low frequency, such cells may exert a disproportion-
ate deleterious effect on tissue physiology by dominant
effects of aberrant cellular regulation (e.g., expression of
inappropriate cytokines) (30). 3) In vitro senescence has
been closely associated with the shortening of telomeric
DNA, at least in humans (56). In human somatic cells, the
average length of telomeric DNA shortens by 50 bp/PD
in vitro, and by 15 bp/yr in vivo (30).
Leaving aside the question of the relevance of replica-
tive senescence, is there any evidence that oxidative dam-
age contributes to it? As early as the mid 1970s, this hy-
pothesis was tested, by culturing human fibroblasts under
conditions of low O2 partial pressures and supplementing
culture medium with the antioxidant a-tocopherol, with
somewhat equivocal results (10, 241, 242). During the
1980s, a handful of studies documented the increased sen-
sitivity of late-passage cells to oxidant challenge (139, 140)
and noted that hyperoxic exposure could shorten in vitro
life span (140). Later studies, looking to PD-dependent
changes in antioxidant defenses as a potential explanation
for the increased sensitivity of late-passage cells to oxi-
dants, failed to account for these observations (250), al-
though an impaired ability of late-passage cells to reini-
tiate DNA synthesis after hyperbaric O2 exposure was
established (141). More recently, a loss of oxo8gua glyco-
sylase activity with increasing passage number has been
reported (133), suggesting that decreased repair may be
involved.
Recently, the issue has been revisited, and it was
shown that a short (2 h) exposure to H2O2 (200 mM) in-
duces an abrupt senescent-like growth arrest in human
diploid fibroblasts (40). Intriguingly, cells that resumed
cell division were found to have a markedly attenuated
life span; a short-term oxidative stress had resulted in a
long-term loss of PDs. The inhibition of H2O2 growth ar-
rest by a metal chelator suggested that a Fenton-derived
jOH radical was involved, and it was proposed that oxida-
tive DNA damage and its sensing by cell cycle checkpoint
controls may be involved in the phenotype (40). Indeed,
measurements of DNA oxidation indicated that senescent
cells suffer greater oxidative DNA damage than young
cells and that the addition of the antioxidant PBN to the
culture medium prolonged the life span in a dose-depen-
dent fashion (41). In these and other studies, it was shown
that culture under low oxygen tension extended fibroblast
life span (41, 355) and that the inhibition of catalase de-
creased it (355), implicating endogenous H2O2 in replica-
tive senescence.
Interestingly, lipofuscin accumulates in fibroblasts in
vitro as well as in vivo, and culture under mildly hyperoxic
conditions (40% O2 partial pressure) decreases life span
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565
and stimulates lipofuscin accumulation (333). Direct feed-
ing of synthetic lipofuscin particles to fibroblasts, which
phagocytose and accumulate the material, simulates se-
nescence (333). Most intriguing, perhaps, is the revelation
that 40% O2 culture resulted in a rapid loss of telomeric
DNA as well as other cytogenetic alterations characteris-
tic of senescent cells, and a permanent loss of replicative
capacity reminiscent of the growth arrest of cells with a
pulse of H2O2 (330a, 334). The measurement of DNA sin-
gle-strand breaks in telomeric sequences of such cells
revealed an increase under hyperoxia (334), confirming
the idea (40) that DNA damage by oxidants may be in-
volved in replicative senescence in vitro and extending
the model to embrace the theory of telomere shortening
(334). Interestingly, it has also been recently shown that
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the repair of ultraviolet light-induced pyrimidine dimers
in telomeric DNA is lower in fibroblasts from an old donor
than a young donor (171).
Together, these experiments are consistent with the
hypothesis that oxidative DNA damage, acting to shorten
telomeric sequences and thereby induce DNA damage-
responsive growth arrest controls (107), plays a role in
in vitro senescence. Two recent studies permit a further
speculation. It has been shown that overexpression of the
oncogenic ras gene in primary cells, which initially serves
as a strong mitogenic stimulus, rapidly induces premature
senescence in a p53- and p16-dependent fashion (286).
Interestingly, it has also been recently reported that ras
induces cellular oxidant generation (147). It is tempting
to suggest that the aggressive mitogenic stimulus (286)
of oncogenic ras, credited with inducing senescence, may
be oxidative in nature. There may be a role for oxidants
in replicative senescence.
XII. CLASSICAL AND POPULATION GENETICS
In comparison to the measurement of age-related
changes, genetic approaches have the potential to provide
more definitive information about aging, by supplying
model organisms (mutant strains with altered life span)
that differ at a small number of loci. Many of the geneti-
cists favorite organisms have been found to be amenable
to studies of aging and have furnished evidence of the
role of oxidants (196, 289).
A. Caenorhabditis elegans Genetics
The emergence of C. elegans as a convenient meta-
zoan genetic model has attracted scientists seeking to
discover genes controlling life span, or genontogenes
(152, 158). Several long-lived mutant strains of C. elegans
have been identified, including age mutants (the originally
identified phenotype being extended life span), daf mu-
tants (identified by abnormalities in the formation of
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566 KENNETH B. BECKMAN AND BRUCE N. AMES
dauer larvae), and clk (clock) mutants [which exhibit a
pleiotropic behavioral deceleration (slower feeding, defe-
cation, and movement)] (158). These two classes of mu-
tant have collapsed into a single category with the cloning
of age-1 and its identification as an allele of daf-23 (219).
Strains of age-1 live 65% longer on average and have a
MLSP 110% longer than wild type (153). The trait is reces-
sive, and biochemical studies have revealed that strains
carrying age-1 alleles have enhanced oxidative defenses.
For example, when the sensitivities of wild-type and age-
1 strains to H2O2 were compared, it was found that the
50% effective lethal dose (LD50) of the wild-type remained
constant, whereas the LD50 of the age-1 mutant increased
with age, an indication of increasing resistance to oxi-
dants (178). Similar comparisons showed that age-1 mu-
tants are more resistant to O20j as well (330). In both
cases, greater resistance to oxidants is associated with
elevated activities of SOD and catalase. Whereas in wild-
type animals these activities remained constant through-
out the life span, in age-1 strains they increased with
advancing age, such that the difference between the mu-
tants and the wild type grew increasingly pronounced at
old ages (178, 330). In addition to being resistant to oxi-
dants, age-1 strains have increased heat tolerance, so it is
likely that enhanced ability to cope with all environmental
stresses, including oxidative ones, is the result of the loss-
of-function mutation (185).
A comparative study of daf-2 and age-1 mutants re-
vealed that they lie in a common pathway. Life extension
by either requires the action of wild-type daf-16, and their
effects on life extension are not additive (72), and so it
was reasoned that they work in tandem. Their cloning re-
vealed that daf-2 is a member of the insulin receptor family
(160a) and that age-1 is a catalytic subunit of phosphatidyl-
inositol 3-kinase (219), suggesting that together they trans-
duce an insulin-like metabolic signal with pleiotropic ef-
fects on metabolism, stress resistance, and dauer formation
(127b, 291a). The more recent cloning of daf-16, revealing
that it is a member of the hepatocyte nuclear factor 3 (HNF-
3)/forkhead family of transcriptional regulators (183a), has
provided insight into the entire pathway, since in humans,
insulin signaling antagonizes HNF-3. The implications,
then, are 1) that signaling through daf-2 and age-1 ulti-
mately may serve to antagonize the control of a genetic
program controlled by daf-16, and 2) that this pathway
may be relevant to human aging, since it appears to have
been conserved (183a). Of course, the connection of these
proteins to the aerobic respiration and the rate of living, via
insulin-mediated metabolic control, has escaped no ones
notice (183a).
The cloning of the clk-1 gene also implicates respira-
tion rate in C. elegans life span, albeit indirectly. The gene
is homologous to a yeast gene called CAT5/COO7 (79a),
which is necessary for the genetic control of the shift from
anaerobic to aerobic metabolism (127b), as well as the
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Volume 78
biosynthesis of the mitochondrial electron transport com-
ponent UQ (252a). It has been proposed that the deceler-
ated metabolic rate in Clk-1 mutants of C. elegans may
result in a correspondingly slower rate of oxidant genera-
tion (127b), although the authors of this work note that so
far data are merely consistent with such a proposal.
A mutant that is in some ways the opposite of age-1
resulted from a screen for sensitivity to O20j in a chemi-
cally mutagenized population of C. elegans. The resulting
mutant, mev-1, is hypersensitive to both paraquat and
hyperoxia. The activity of SOD in mev-1 was found to be
roughly one-half of wild type, and the average life span
was reduced by 35% (148). Lipofuscin-like fluorescence
accumulated more rapidly in mev-1 mutants that in wild-
type animals (144). The life span of both mev-1 and wild-
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type nematodes was lengthened under hypoxia and short-
ened by hyperoxia, but the effect was more dramatic for
the mutants (138).
B. Drosophila Genetics
According to the evolutionary concept of antagonis-
tic pleiotropy, natural selection favors traits that benefit
organisms during their period of reproduction. Therefore,
by imposing a strong artificial selection that consistently
rewards females capable of late reproduction, one ought
to be able to select against those pleiotropic traits that are
most antagonistic to long-term survival. After a suitable
number of populations, a long-lived phenotype should
emerge from a large, heterogeneous population.
In the early 1980s, Rose (264) followed precisely this
strategy using Drosophila thereby validating the concept
of antagonistic pleiotropy and also providing five indepen-
dently selected lines (referred to as lines O1-O5) for fur-
ther experimentation. The strength of this model is that,
unlike age-1, lines O1-O5 are not single gene mutants, but
rather populations in which any number of allelic changes
will have occurred. Hence, biochemical alterations that
are found to have arisen independently in many of these
lines are likely to reflect the type of complex polymor-
phisms that confer increased life span to long-lived spe-
cies such as Homo sapiens.
Therefore, it is significant that in a comparison of the
lines O1-O5 with lines B1-B5 (control lines not subjected
to selection), all of the long-lived populations had ac-
quired a higher frequency of a relatively rare SOD allele
that is associated with higher in vitro activity (328). In
Drosophila, there exist two electrophoretic alleles of
SOD, designated F (with lower in vitro activity) and S
(with higher in vitro activity). Whereas the genotypes of
all B flies tested were FF (no S alleles were detected),
both FS and SS individuals existed in all five O lines, such
that the average allele frequency of S ranged from 0.10 to
0.28 [0.25 { 0.05 (SE)]. In all five independent instances
of laboratory evolution, SOD activity had increased.
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April 1998 FREE RADICAL THEORY OF AGING
C. Rodent Genetics
The senescence-accelerated mouse (SAM) is an in-
creasingly important model in gerontology (244); the se-
nescence-prone strain (SAM-P) has a mean life span of 9
mo, compared with 13 mo for a senescence-resistant
(SAM-R) strain. In liver tissues, the activity of mitochon-
drial SOD in the SAM-P strain was about one-half that in
the SAM-R strain, a result that was consistent at all ages
examined (244), which could explain the more rapid age-
related increase in lipid peroxidation seen in this tissue
(244) as well as independent reports of enhanced oxida-
tive stress in SAM-P mice (186).
A potentially very useful animal model for testing the
free radical theory of aging is the S strain of Wistar rat,
selected for sensitivity to cataractogenesis by galactose.
A heritable increase in cellular hexose uptake by cells
from the S strain is associated with an increased intracel-
lular generation of oxidants, increased endogenous lipid
peroxidation, mitochondrial dysfunction, an increased
age-specific incidence of degenerative diseases, and ear-
lier aging than the galactose-resistant R strain (271, 272).
Moreover, SOD and catalase activities in the blood of S
rats are one-half that in R rats, which may have explained
the increased in protein oxidative damage that was also
seen in the S strain (345).
XIII. MOLECULAR GENETICS
A. Oxidants and Nuclear Somatic Mutations
Oxidants are twice removed from the somatic muta-
tion theory of aging, since two requirements need to be
satisfied for the relevance of oxidants to be ensured: 1)
oxidants must account for a significant proportion of mu-
tations, and 2) mutations must account for a significant
proportion of the phenomena of aging. The second crite-
rion is complex and has been recently discussed (71a,
195, 218, 338b); suffice it to say that to the extent that
cancer is a degenerative disease of age (6), there is little
doubt that mutations are relevant. What remains to be
seen is whether or not the age-related degenerative
changes that do not involve neoplasia are also the result
of mutations (79) (or epigenetic alterations; Ref. 136).
With the assumption that they are, the question then
remains, To what extent are spontaneous mutations due
to oxidants? Some of the best evidence so far of the rele-
vance of oxidative mutagenesis in vivo is an argument by
analogy. In E. coli, mutation rates can be elevated from
10- to 1,000-fold by the loss of mutator genes mutM, mutT,
and mutY, whose gene products are involved in repair
and prevention of oxidation of guanine in DNA (210).
Significantly, homologous genes and novel genes with an-
alagous activities have been found in a number of species
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567
including humans (17, 192, 266), which forcefully makes
the case that oxidants are significant mutagens in vivo.
There is, moreover, plentiful direct evidence of oxidative
mutagenesis in eukaryotic cells, such as the demonstra-
tion that disruption of the metallothionein gene in tissue
culture, which may abrogate the cells ability to chelate
redox active metals such as iron, results in a 5- to 10-fold
increase in spontaneous mutagenesis (267). Also, muta-
genesis in cells from humans with Fanconis anemia, a
condition associated with oxidative stress, displayed a
higher mutation frequency than controls and a marked
increase in mutation frequency in response to the eleva-
tion of O2 partial pressure (183).
Despite these results, the proportion of mutations
due to oxidants in the presence of wild-type antimutators
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is still an unknown quantity. Recent experiments have
illustrated that dietary supplementation with antioxidants,
which reduces irradiation-induced mutagenesis, appears
to reduce the spontaneous mutation rate as well (99, 100),
suggesting that antioxidant scavenging in vivo is incom-
plete. The quantitative contribution of oxidants in somatic
mutagenesis is an open (and important) question.
B. Oxidants and Mitochondrial Somatic Mutations
A burst of activity was stimulated by the report that
a large deletion of the mitochondrial genome (DmtDNA)
accumulates exponentially with age in humans and by the
speculation that this may be the work of mitochondrial
oxidants (53). Since this first detection of DmtDNA in
normal elderly humans, dozens of studies have confirmed
the finding, detecting the same common deletion in hu-
mans as well as different deletions in other species, in-
cluding C. elegans (206), mice (44, 323), rats (76), and
rhesus monkeys (179). Increased frequencies of DmtDNA
in brain samples have been associated with Huntingtons
disease and AD, degenerative diseases of aging that affect
millions of people (48, 143).
In most of these cases, a significant accumulation of
the deletions occurs only at advanced ages and is associ-
ated primarily with postmitotic tissues. This mosaicism
of mtDNA deletion (313), which is reminiscent of the pat-
tern the maternally transmitted mitochondrial diseases, is
hypothesized to be partly due to the lack of proliferative
competition in postmitotic tissues (50, 336). Whereas in
dividing tissues cells that inherit a relatively greater per-
centage of DmtDNA molecules by unequal mitotic assort-
ment stand to compete less well in subsequent rounds of
cell division, this phenotypic sieving will not operate in
a nondividing tissue.
There are reasons to believe that the deletion of
mtDNA may stem from oxidative damage (51). For one, the
steady-state burden of oxidative adducts in mtDNA has
been measured to be 16-fold higher in rat mtDNA than in
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568 KENNETH B. BECKMAN AND BRUCE N. AMES
nuclear DNA (16, 260). Moreover, quantitative polymerase
chain reaction analyses of DmtDNA accumulation in differ-
ent areas of the brain have consistently revealed highest
frequencies of areas of high metabolic activity (47, 54). De-
spite the promise of these results, it is not self-evident that
the observed accumulation of DmtDNA represents more
than a biomarker (182). For one, the highest frequencies of
DmtDNA detected, even at the oldest ages and in the most
deletion-prone tissues, are generally no higher than 0.1%.
Because in maternally transmitted mitochondrial genetic
disorders symptoms are often absent even when more than
one-half of the mtDNA molecules carry deletions (336), it
is hard to see how such low frequencies could exert an
effect. On the other hand, because there may be numerous
deletions existing independently, measurements of a single
one may merely represent the tip of the iceberg of mtDNA
damage (54). The use of long polymerase chain reaction
to amplify the entire mitochondrial genome has revealed the
presence of numerous mtDNA rearrangements in human
skeletal muscle (207). On the other hand, a recent attempt
to measure an increase in somatic point mutations in human
skeletal muscle was unsuccessful (243).
The analysis of progressively smaller bundles of cells
has shown that the DmtDNA frequency is unevenly dis-
tributed (282). For example, when a muscle homogenate
of rhesus monkey fibers is analyzed, the detected fre-
quency of DmtDNAs is 0.020.1%. However, restriction
of the sample size to 10-fiber bundles decreases the num-
ber of positive reactions but increases the measured fre-
quency of DmtDNAs in positive reactions to 4.613.2%.
In other words, a frequency of 0.1% could represent 100%
deleted molecules in 1 cell of 1,000 (180). At any given
point in time, only a small number of cells may carry
deletions, but if these cells continually die as a result of
a failure of oxidative phosphorylation, and other deletions
arise de novo, then the low steady-state frequency of dele-
tions could obscure a high rate of DmtDNA-associated
cell death.
Finally, it has been proposed that a vicious cycle of
increasing oxidative damage and stress of the type dis-
cussed in section IIF is particularly likely in the case of
mtDNA, since it encodes components of the mitochon-
drial electron transport chain (52). This hypothesis is sup-
ported by the fact that in statistical terms, a random muta-
tion of mtDNA is most likely to affect complex I, com-
bined with the observation that a complex I poison
(MPTP) triggers neuronal cell death in mice (52). It re-
mains to be shown that known somatic mtDNA mutations
do, in fact, result in increased oxidant generation.
XIV. TRANSGENIC ORGANISMS
A. Transgenic Drosophila
The modern acid test of a biological theory is the
generation of a transgenic organism, and recent efforts to
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genetically engineer long-lived Drosophila by bolstering
antioxidant defenses have been successful. The first such
efforts involved the introduction of a bovine cDNA for
Cu,Zn-SOD into Drosophila, which resulted in a signifi-
cant increase in SOD activity as well as resistance to oxi-
dative stresses and a small, statistically significant in-
crease in mean life span (85). Whereas a repeat of this
procedure with the Drosophila Cu,Zn-SOD gene resulted
in only marginal effects on resistance to oxidants, and
failed to increase life span, the introduction of single cop-
ies of Drosophila SOD and catalase resulted in a number
of strains with significantly extended mean and maximum
life spans (237).
As expected, the augmentation of antioxidant activi-
ties in the transgenic flies led to significant improvements
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over controls in numerous age-related indexes of oxida-
tive metabolism: 1) the accumulation of protein carbonyls
was reduced; 2) the rate of oxygen consumption at middle
age and old age was higher; 3) the 50% loss of G-6-PD
activity with age was reduced to 10%; 4) the age-related
doubling of the load of oxo8dG was virtually abolished,
as was the roughly twofold increase in sensitivity to the
induction of oxidative DNA damage; and 5) the age-re-
lated threefold increase in mitochondrial oxidant genera-
tion was reduced by about one-half (237, 298). In short,
the predictions of the free radical theory were confirmed.
B. Transgenic Mice
Ultimately, transgenic mice are the molecular geron-
tologists Holy Grail, and it is not until similar experiments
are performed in mice that the free radical theory will
have been satisfactorily tested. Although such experi-
ments will clearly be exciting, work that has already been
performed with mice transgenic for SOD suggests that
their phenotypes may not be straightforward. As has been
stressed, antioxidant defenses are interactive and coordi-
nately regulated, and it has become apparent that oxidants
play important roles as signaling molecules (286). As a
consequence, up- or downregulating a single antioxidant
may disrupt an internal balance and either exacerbate
oxidative stress or otherwise compromise cellular physi-
ology. Indeed, the phenotype of Cu,Zn-SOD-overex-
pressing mice appears to mimic the pathology of Downs
syndrome (for which they may be a model, due to the
localization of Cu,Zn-SOD to the trisomic chromosome
21; Ref. 36). Moreover, it has been recently discovered
that a complete knockout of Cu,Zn-SOD has a phenotypic
effect only on a small subset of motor neurons thought
to be involved in amyotrophic lateral sclerosis (255). Even
more surprising, the recent elimination of the enzyme
GPX in a transgenic knockout mouse model resulted in
generally normal, fertile mice without overt signs either
of oxidative stress or increased sensitivity to oxidants
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April 1998 FREE RADICAL THEORY OF AGING
(134). Together, these results indicate that the promising
results with Drosophila may be difficult to replicate, al-
though no matter what the results, antioxidant overex-
pressing strains will clearly be extremely useful. By cross-
ing animals with different transgenes, it will be possible
to dissect antioxidant interactions and their effects on
aging.
XV. SPORADIC DEGENERATIVE DISEASES
In the form of the specific age-related diseases in
elderly humans, there exists a natural laboratory of oxida-
tive stress. Indeed, investigation of the etiologies of hu-
man degenerative disease implicate oxidative stress in
cancer (5, 6), atherosclerosis (112, 343), diabetes (326),
and neurodegeneration (15, 18, 293), to name a few.
For example, the role of oxidants in AD has been a
particularly active area of research in recent years. It has
been known for some time that the formation of amyloid
deposits, a hallmark feature of the clinical diagnosis, is
associated with aberrant processing and folding of a pro-
tein called amyloid b-precursor protein (AbPP) to form
the amyloid plaques (Ab) characteristic of the disease
(283). In recent years, it was discovered that not only
the in vitro formation of synthetic Ab from AbPP can be
accelerated by the presence of oxygen but also that Ab,
which is cytotoxic when fed to neurons in vitro, appears
to stimulate the generation of oxidant (293). In fact, amy-
loid peptides themselves generate oxidants in vitro (130).
As discussed above, it has also been recently discovered
that a cellular receptor (on microglial cells and endothe-
lial cells) responsible for transducing the cytotoxic signal
from Ab and inducing oxidant generation is none other
than RAGE (344). Amyloid plaques have also been found
to bind redox-active iron, providing another potential pos-
itive feedback loop for oxidative damage (292a). Although
we do not have space to go into the details, what is rele-
vant here is not so much the specifics but the realization
that in numerous ways, oxidants act as effectors in the
progression of the disease. In other words, research into
the etiology of AD (193b, 203a, 292b) [and also Parkinsons
disease (15, 44a, 145a), amyotrophic lateral sclerosis
(346), and dozens of other age-related disorders] is reveal-
ing that cytotoxicity very frequently involves oxidative
stress.
Even if oxidants turn out to be essential effectors of
AD pathology, can they initiate the process? Recently, it
was claimed that a higher degree of heteroplasmy of
mtDNA is associated with individuals with AD versus con-
trols (62), consistent with a genetic predisposition based
on mitochondrial inheritance. What is intriguing in this
work is the reported association of these AD polymor-
phisms with higher intracellular oxidant generation by
mitochondria isolated from the patients (62). However,
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569
recent work by two groups has independently revealed
an artifact in the unusual protocol used for mtDNA extrac-
tion in these experiments: a nuclear pseudogene, rather
than mtDNA itself, was amplified, and so the conclusions
from these studies are invalid (131b, 336a).
XVI. INHERITED DEGENERATIVE DISEASES
Werners syndrome (WS) is a segmental progeroid
syndrome of late-onset accelerated aging, associated with
an increased incidence of cancer, attenuated replicative
senescence of cells in vitro, and, interestingly, an eleva-
tion of oxidized protein in isolated fibroblasts (315, 354).
The cloning (354) of the gene responsible for the syn-
drome (WRN) does not suggest an oxidative etiology,
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however, as the protein product WRN appears to be a
helicase (106a). WRN is homologous to the RecQ helicase
of E. coli, which is involved in unwinding DNA during
repair; hence, it has been proposed that the loss of heli-
case activity in WS results in a deficiency in recombina-
tion-mediated repair in humans cells (106a, 291a). Ineffi-
cient or dysregulated DNA repair, in turn, may cause the
chromosomal instability, which is one of the diseases
hallmarks and which may be the root of some or all of
the diseases associated with the syndrome (106a, 291a).
Although there is consequently no obvious causal link
between mutations in WRN and oxidative damage, the
increased protein oxidation in WS fibroblasts (315) allows
the speculation that measurable oxidative damage may
result from inefficient recombination-dependent repair,
chromosomal instability, and an ensuing disruption of ge-
netic programs. If this turns out to be the case, the oxida-
tive phenomenology of WS will serve as an important
reminder that the measurement of oxidative damage does
not imply oxidative causation! [On the other hand, if nor-
mal aging, like WS, results in part from chromosomal in-
stability, it may be that oxidative genotoxicity plays a
central role (sect. XIIIA). In other words, it may be that
oxidative damage may both result from and result in chro-
mosomal instability.]
XVII. EPIDEMIOLOGY OF OXIDANTS
AND ANTIOXIDANTS
Nutritional epidemiology has suggested that dietary
antioxidants are crucially involved in the prevention of
degenerative disease (22), and global epidemiological
studies have pointed to geographical and economic anti-
oxidant deficiencies in their localized occurrence (6). For
instance, the loss of immune function with aging is amena-
ble to therapeutic treatment with antioxidants (208a); the
fact that both antioxidant status and immunological de-
cline vary between individuals (209) suggests that there
may exist heritable difference in antioxidant capacities,
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570 KENNETH B. BECKMAN AND BRUCE N. AMES
linked to differences in immune function. To date, how-
ever, the epidemiological study of senescence is merely
a theoretical possibility and awaits powerful tools before
it can be affordably attempted.
XVIII. SUMMARY
A. Are Oxidants Responsible for Aging?
We have outlined what we see to be the major experi-
mental approaches to testing the free radical theory of
aging. In Table 1, the strengths and weaknesses of differ-
ent modes of experimentation are assessed. How power-
ful are different types of experiment? Has the free radical
theory been supported?
Before a discussion of the merits of individual experi-
ments, it should be stressed that if one is to judge them
by their abilities to falsify the free radical theory, they are
all bound to fail, for the simple reason that the free radical
theory is very hard to falsify. The absence of a predicted
outcome (for instance, life span extension by antioxidant
supplementation) may often be explained by (justifiable)
ad hoc reasoning. The physiology of oxidative stress, be-
ing a complex interaction of endogenous and exogenous
factors, generally permits the explanation of negative drawback of such experiments is that they have generally
results. How then shall one judge the different types of
experiments and the theory itself? In fact, as the results
discussed above illustrate, the momentum gathering be-
hind the free radical theory is not due to any single experi-
ment or approach, but rather derives from the extraordi-
narily multidisciplinary nature of current research. Al-
though no single line of reasoning alone permits definitive
conclusions, together they present a compelling case.
[This philosophy, that the study of aging should employ
a combination of different approaches, has been convinc-
ingly espoused before (83).]
Age-related oxidative phenomenology continues to
provide evidence that senescence is associated with in-
creased oxidant generation, a decline in the robustness
of defenses and repair, and an accumulation of end prod-
ucts of oxidative damage (sect. IV). Although these trends
are only correlations, the study of age-related changes has
been rewarded with surprises such as the receptor for
AGE (RAGE), which turns out to be involved in the etiol-
ogy of AD and evidence that lysosomal lipofuscin may
directly disrupt cells in vivo and in vitro. In fact, oxidative
phenomenology has focused efforts on understanding the
interplay between the components of oxidative stress out-
lined in Figure 1. The search for evidence of age-related
DNA and protein oxidation has stimulated research into
DNA repair, proteolytic salvage pathways, mitochondrial
defenses, and so on.
Interspecies comparisons have better potential di-
rectly to test the free radical theory (sect. V). As predicted,
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Volume 78
differences between long- and short-lived species have
been documented in all components of oxidative stress.
Perhaps most instructive, however, have been results that
initially appeared contradictory, such as the fact that birds
are an outlier group in rate of living calculations. Attempts
to account for the discrepancy have led to promising ad
hoc hypotheses about the role of mitochondrial oxidant
generation in aging. Unfortunately, relatively few re-
searchers are familiar with or prepared to handle a variety
of experimental animals, despite the research potential of
a diverse menagerie.
In contrast, the model of dietary restriction has ap-
peal precisely because it is so well established, homoge-
neous, and relevant to specific human diseases (sect. VI).
Although early predictions that DR would result in a lower
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metabolic rate have proved unfounded (312), the discov-
ery that oxidative defenses are enhanced by DR have
strengthened both the free radical theory and the concept
of antagonistic pleiotropy (158). Although, currently, re-
sults from DR studies are merely consistent with the free
radical theory, future experiments may reveal specific
pathways whereby DR upregulates defenses and repair.
Manipulation of metabolic activity and oxygen con-
centration are interesting systems, if of somewhat ques-
tionable relevance to normal aging (sects. VII and VIII). One
been limited to invertebrates; the manipulation of metabo-
lism in mammals is less straightforward. In the future,
transgenic mouse models may allow a specific manipula-
tion of metabolic rate in a consistent genetic background.
Attempts to manipulate life span with nutritional and
pharmacological antioxidants obviously hold great ap-
peal, as direct and intuitive tests of the theory (sects. IX
and X). Although until recently they have not quite lived
up to their promise (at least in mammals), this may have
been due to a lack of the appropriate compounds. The
serendipitous discovery of PBNs pharmacological prop-
erties appears to have opened up new avenues.
The study of fibroblast aging in vitro might appear
the least likely area of research to support the free radical
theory, being a phenomenon of replication per se, rather
than of chronological aging (sect. XI). Nevertheless, the
results discussed above not only suggest that oxidants
may play a role, but they also have led to novel hypotheses
about the dynamics of telomeric DNA in vivo. Future work
in this area should be exciting.
The utility of genetics and transgenic animals in the
study of aging, on the other hand, does not need elabora-
tion (sects. XII and XIV). At present, the chief limitation to
genetic studies is that fact that the adult forms of C. eleg-
ans and Drosophila are postmitotic. Molecular genetics
(sect. XIII), which in the study of aging has primarily cen-
tered around the concept of somatic mutagenesis, is a
field with particularly powerful tools that are generally
applicable to any species. It is therefore very well suited to
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April 1998 FREE RADICAL THEORY OF AGING
comparative studies, as illustrated by the hunt for mtDNA
deletions in a wide range of species. Moreover, because
mutational spectra provide information about the primary
mutagenic event (256), molecular genetic studies may find
the smoking gun of oxidative mutagenesis.
Finally, the push to understand human sporadic and
inherited diseases through biochemical, genetic, and epi-
demiological methods may ultimately provide the most
detailed information about age-related oxidative stress, as
is becoming apparent in the cases of cancer, atherosclero-
sis, and neurodegenerative disorders (sects. XV XVII). The
complete sequencing of the human genome, and modern
high-throughput genetic methods, are poised to provide
an unprecedented amount of genetic information about a
biological sample of many millions of individuals whose
life histories (medical records) are accessible. For in-
stance, an approach to the genetic study of aging that is
only now attracting mainstream interest is the study of
human longevity outliers, or centenarians (292). Techni-
cal advances in the identification and analysis of polymor-
phisms, fueled by the Human Genome project, may permit
the study of oxidative stress-associated alleles in these
extraordinary individuals. An example of such an ap-
proach is the identification of a coding mtDNA polymor-
phism (a C-to-A transversion at mtDNA nucleotide posi-
tion 5178 in the NADH dehydrogenase subunit 2 gene) in
a larger percentage of centenarians (62% with 5178A) than
controls (45% with 5178A) (322a). In this study, it was
also found that as the age of randomly selected hospital
inpatients and outpatients increased, the percentage car-
rying the 5178C polymorphism increased, suggesting that
such individuals are at higher risk of degenerative disease.
The authors hypothesize that the 5178A polymorphism,
which is rare in all human populations except the Japa-
nese, may be responsible both for the longevity of Japa-
nese centenarians and, therefore, for the general longevity
of the Japanese population as a whole. They suggest that
such a polymorphism, affecting the mitochondrion, may
have an effect on oxidative stress.
The answer to the question of whether or not the free
radical theory has yet been confirmed depends on ones
conception of what the theory predicts. In its broader
sense (oxidants contribute significantly to the process of
degenerative senescence), the theory has clearly been
validated. In the more strict sense of the theory (oxidants
determine MLSP), the data are not yet conclusive, al-
though a large body of consistent data has been generated.
B. Nitric Oxide
A topic of great importance, which we have here
largely omitted, is the role of nitric oxide (NOj) both as
a damaging oxidant in its own right and as a signaling
molecule that is susceptible to oxidative scavenging. NOj,
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571
the chemical previously known as endothelium-derived
relaxation factor, is generated enzymatically by isozymes
of nitric oxide synthase and is involved in vascular tone,
innate immunity, and neuronal signal transduction. More-
over, NO reacts with O20j to form peroxynitrite (ONOO0),
itself a powerful oxidant. Therefore, not only must nitric
oxide synthase be seen as a potential source of damaging
oxidants (249, 281), but O20j may now be considered a
physiologically relevant scavenger of the free radical sig-
naling molecule NOj (58).
C. Oxidants and Apoptosis
Another topic we have decided to omit, because of
space limitations and because this is an extremely fast-
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moving and frequently reviewed area, is the role of oxi-
dants and mitochondria in apoptosis. Suffice it to mention
that in the previous year there have appeared dozens of
experiments documenting either the loss of mitochondrial
membrane potential (355a), the release of mitochondrial
cytochrome c (255a) [or an apoptosis inducing factor
(355a)] into the cytosol, or the generation of oxidants by
mitochondria during apoptosis (355a). Combined with the
mitochondrial localization of the Bcl-2 family of anti-
apoptosis proteins, and copious evidence that oxidant
challenges can induce apoptosis (74a, 154, 290a), these
experiments have shown that mitochondria are central to
the process of cell suicide (255a, 355a). Despite this gen-
eral consensus, however, a number of disagreements exist
about the details of the mitochondrions role in the pro-
cess (149a) and even greater uncertainty about the role
of oxidants. An equally complex problem will be defining
the role of apoptosis itself in aging (338a). For instance,
by eliminating neuronal cells, apoptosis may contribute to
neurodegeneration, yet effective apoptosis is also clearly
critical in preventing (age-related) tumorigenesis. In
short, both the machinery and impact of apoptosis are still
incompletely understood; conclusions about the interplay
among oxidants, apoptosis, and aging will have to await
further experimentation (338a).
D. Regulatory Roles for Oxidants and
Antioxidants: Signal Transduction
The discovery that NOj could serve as a signaling mol-
ecule has foreshadowed a more general recognition that
radical species are more than by-products. As mentioned
in section XVIIIB, the interaction between O20j and NOj
indicates that both may be regulatory molecules (58). In
other ways, the relationship between oxidants and signal-
ing is surfacing. For instance, the nitration of tyrosine resi-
dues on a synthetic peptide substrate of cell cycle kinases
destroyed its ability to be phosphorylated, which illustrates
a mechanism for an individual amino acid adduct to exert
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572 KENNETH B. BECKMAN AND BRUCE N. AMES
an amplified effect (167). At a more physiological level, it
has been shown that the sensitivity of hippocampal brain
slices to muscarinic acetylcholine receptor agonists, which
falls in an age-related fashion, can be restored by various
antioxidant treatments and is potentiated (in old animals)
by NOj-generating systems (155).
Perhaps most intriguing have been discoveries, such
as the potential regulation of aconitases by O20j (95, 98)
and the realization that cell signaling via the Ras family
of tyrosine kinases involves oxidants (147), that suggest
that oxidants play a role in signal transduction by design.
These discoveries strengthen the free radical theory for
the following reason: if the regulated, enzymatic genera-
tion of oxidants plays a role in signaling pathways (legiti-
mate oxidants), then even a small increase in oxidant
generation by nonregulated pathways (illegitimate oxi-
dants) may have an exaggerated impact, by mimicking
the legitimate process. The arrival of redox regulation
as a viable field of inquiry, as evidenced by numerous
topical recent reviews (66, 285, 321), may usher in a period
when oxidants are seen as molecules of central physiolog-
ical importance, in much the same way as are (for in-
stance) lipid metabolites. As far as acceptance of the free
radical theory is concerned, such a paradigm shift will
probably lead to de facto acceptance of its broader con-
ception (the idea that oxidants play a role in the process of
aging). After all, if it turns out that oxidants and oxidative
reactions are central to physiology, then how could they
not be central to aging as well? If the free radical theory
once lacked appeal because oxidants appeared to be stoi-
chiometrically minor by-products, then those days may
soon be over.
On the other hand, in its more narrow conception
(oxidants define MLSP), the free radical theory still
lacks experimental confirmation. A hypothetical example
will illustrate this point. Even if mitogenic stimulation by
Ras, for instance, were to involve oxidant generation in
mice and humans, and the disregulation of Ras signaling
by illegitimate oxidants in aged individuals were shown
to be important in degenerative senescence, the different
life spans of these organisms still beg an explanation; it
still would remain to be explained why the process of
oxidative disregulation takes so much longer to occur in
humans than in mice. In conclusion, despite the growing
concensus that the oxidants and oxygen free radicals are
involved in degenerative senescence, countless mechanis-
tic questions remain to be uncovered, as well as a central
outstanding unknown: Do oxidants determine maximum
life span in humans and other organisms?
We thank Caleb Finch for critically reading the manuscript
and Irwin Fridovich for offering numerous detailed and useful
comments. K. B. Beckman thanks Dr. I. Philipson for numerous
useful discussions.
B. N. Ames was supported by National Cancer Institute
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Volume 78
Outstanding Investigator Grant CA39910 and National Institute
of Environmental Health Sciences Grant ES-01896.
In writing a review on as broad a topic as the free radical
theory, we have been forced to limit both the content and the
number of references cited. Although we have done our best to
include recent work, omissions were inevitable. We apologize
to all authors whose work we have not managed to include and
direct readers to other recent reviews for material we have left
out.
Address for reprint requests: K. B. Beckman, Dept. of Mo-
lecular and Cell Biology, 401 Barker Hall, Univ. of California,
Berkeley, CA 94720-3202.
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